Palytoxin: a Palytoxin Prodrug That Can Be Activated by a Monoclonal Antibody-Penicillin G Amidase Conjugate1

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[CANCER RESEARCH 52. 5759-5764, October 15. 1992] jY-(4'-Hydroxyphenylacetyl)palytoxin: A Palytoxin Prodrug That Can Be Activated by a Monoclonal Antibody-Penicillin G Amidase Conjugate1

Gary S. Bignami,2 Peter D. Senter, Paul G. Grothaus, Keith J. Fischer, Tom Humphreys, and Philip M. Wallace

Hawaii Biotechnology Group, Inc., Aiea, Hawaii 96701 [G. S. B., P. G. G., T. H.f, and Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, Washington 98121 Â¡P.D. S.. K. J. F.. P. M. W.J

ABSTRACT include prodrugs of agents that act at the level of the cell surface and have toxicities that are too great for systemic administra Palytoxin (PTX), one of the most toxic nonprotein molecules known, tion. This might successfully address the potential limitations is cytotoxic at picomolar concentrations against a wide variety of cell types. In contrast to most cytotoxins, PTX exerts its activity extracel- of low drug potency and poor conjugate localization. An addi lularly. A method for targeting PTX to tumor cells is described in which tional advantage may be gained by releasing drugs that are not a monoclonal antibody-enzyme conjugate activates a PTX prodrug at subject to normal multidrug resistance pathways. surfaces of tumor cells. The prodrug, /V-(4'-hydroxyphenylacetyl)paly- PTX (Fig. 1), which is obtained from tropical coelenterates toxin (NHPAP), was prepared by reacting PTX with an active ester of (soft coral) of the genus Palythoa, is one of the most potent 4-hydroxyphenylacetic acid. NHPAP was 1000 times less toxic than cytotoxins known (20, 21). The toxin acts extracellularly by PTX to a panel of carcinoma and lymphoma cell lines. The cytotoxic binding to a cell surface receptor and creating a pore in cell activity of the combination of penicillin G anÃºdasefrom EscherÃ¬chiacoli membranes (22). This is achieved after PTX binds (Kt, 2 x with NHPAP was equal to PTX. Two cell lines that were multidrug 10~" M)to Na^-K^-ATPase, an enzyme that maintains trans- resistant showed no enhanced resistance to NHPAP Â±penicillin G membrane Na+ and K"1"gradients in mammalian cells (23, 24). amulase. Immunologically specific activation of NHPAP took place when H2981 cells (L6 antigen positive) were treated with the mono Picomolar concentrations of PTX are cytotoxic in vitro against clonal antibody conjugate L6-penicillin G amidase followed by NHPAP. cultures of lymphocytes, fibroblasts, and normal or virus trans This system is distinguished from other prodrug activation schemes, formed epithelial cells (25-27). PTX also depolarizes and lyses since the released drug exerts its activity extracellular!}, has high po mammalian erythrocytes (21, 28). Therefore, PTX appears to tency, and may be able to overcome the multidrug resistant phenotype. kill cells that express the Na+-K+-ATPase associated toxin re ceptor, as opposed to most anticancer drugs which preferen INTRODUCTION tially intoxicate metabolically active cycling cells. Because of Efforts to overcome the inherent lack of specificity of anti- this, there may be significant advantages in generating PTX cancer drugs using mAb3 as drug delivery agents have been with mAb-enzyme conjugates, compared to conventional chemotherapeutics. largely unsuccessful in the clinical treatment of solid tumors (1, It has previously been reported that W-acyl PTX derivatives 2). This is most likely due to the very small proportion of the mAb-conjugate that localizes within tumor masses (3), the het are at least 100 times less potent than PTX (26, 29). This provided us with the rationale for developing other TV-acylPTX erogeneity and sometimes widespread distribution of tumor as derivatives that might serve as prodrugs for targeted enzymes. sociated antigens on normal tissues, and the difficulties that We have prepared NHPAP as a substrate for EscherÃ¬chiacoli arise in releasing the targeted agents intracellularly where they PGA. Cytotoxicity studies indicate that NHPAP is up to 1000 exert their cytotoxic activities (4, 5). While it might be possible to circumvent these limitations by delivering highly cytotoxic times less cytotoxic to a number of tumor cell lines than PTX drugs that act extracellular!)', little attention has been focused and that PGA catalyzes the conversion of NHPAP to PTX. PGA was covalently attached to the L6 mAb (anti-carcinoma). in this direction. We, and others, have recently described a strategy in which It was found that the conjugate could effectively activate NH mAb-enzyme conjugates that are bound to tumor associated PAP in an antigen dependent manner on cells that bound the mAb-PGA conjugate. Site-specific generation of highly toxic antigens convert relatively nontoxic drug derivatives (prodrugs) into active anticancer drugs (6-19). The prodrugs reported for molecules such as PTX may constitute a powerful approach for this targeting strategy are either converted to approved antican- chemotherapy with targeted enzymes. cer drugs or to drugs that are closely related to those used clinically. All of the released drugs have intracellular sites of MATERIALS AND METHODS activity. It should be possible to extend the methodology to Materials Received 5/12/92; accepted 8/8/92. The costs of publication of this article were defrayed in part by the payment of PTX was isolated from Hawaiian Palythoa tuberculosa according to page charges. This article must therefore be hereby marked advertisement in accord published methods (20, 25). Purified PTX standards (>95%) were ance with 18 U.S.C. Section 1734 solely to indicate this fact. stored at -20Â°C in 50% aqueous ethanol. PTX concentration and pu 1This publication was supported in part by Grant 1R43CA54652-01 from the rity were assayed by UV spectroscopy between 300 and 190 nm (<233= National Cancer Institute (G. S. B.). Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Cancer 40.500; Ã26Ã=23,600) (30). E. coli PGA (31 units/mg with penicillin G Institute. as the substrate) was obtained as an ammonium sulfate suspension from 2 To whom requests for reprints should be addressed, at Hawaii Biotechnology Group, Inc., 99-193 Aiea Heights Dr., Aiea. HI 96701. Boehringer Mannheim, GmbH. The L6 mAb (IgG2a). which binds to 'The abbreviations used are: mAb. monoclonal antibody: NHPAP, A'-(4'-hy- an antigen on human carcinomas, was isolated as previously described droxyphenylacetyOpalytoxin; ICso, 50% inhibitory concentration; PGA. penicillin (31). The cell lines used were H2981 [human lung adenocarcinoma G amidase: PTX, palytoxin; SMCC, succinimidyl 4-(.V-maleimidomethyl)cyclo- (31)1, LX-1 [human oat cell carcinoma (32)], H3719 [established at hexane I-carboxylate; d. doublet: m. multiplet; LFMEM, leucine-free minimum Bristol-Myers Squibb, Seattle, WA, from a human colon carcinoma], essential medium supplemented with 10% fetal bovine serum, 2 m\t L-glutamine, and 1 mvi sodium pyruvate; IMDM, Iscove's modified Dulbecco's medium with H3639 [established at Bristol-Myers Squibb from a human ovarian 10% (v/v) fetal calf serum. 200 units/ml penicillin, and 0.1 mg/ml streptomycin; carcinoma], HSB-2 [human T-cell lymphoma; American Type Culture NMR, nuclear magnetic resonance. Collection CCL 120.1], EL4 [murine T-cell lymphoma; American Type 5759

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Fig. 1. Structures of PTX and NHPAP.

PTX NHPAP

Culture Collection TIB 39]. HCTI16 and HCT116(VM)46 (human trobenzoic acid) as previously described (15). Under these conditions, colon carcinomas (33)], and A2780 and A2780DDP [human ovarian 1-2 maleimides were incorporated per PGA molecule. carcinomas (34)]. Reduction of native disulfides present in the L6 mAb was achieved by treating L6 (9-11 mg/ml in 10 RIMsodium phosphate, pH 7.2, con Synthesis of NHPAP taining 0.15 MNaCl) with dithiothreitol (final concentration, 1 RIM)at 23Â°Cfor 15 min. The antibody was separated from low molecular To a solution of 4-hydroxyphenylacetic acid (107 mg, 0.7 inmol) in tetrahydrofuran (7 ml) at 23Â°Cunder an argon atmosphere was added weight materials on a Sephadex G-25 column using 40 RIMsodium A'-hydroxysuccinimide (81 mg, 0.7 mmol) and AVV'-dicyclohexylcarbo- phosphate containing 500 mMNaCl at pH 7.2 as eluant. The number of free thiol groups (2-3 per L6 molecule) was determined using 5,5'- diimide (144 mg, 0.7 mmol). After l h of stirring, 0.37 ml (theoretically dithiobis(2-nitrobenzoic acid) as previously described (15). containing 37 mmol of the active ester) of the supernatant was trans The modified proteins were combined in a 1:1 molar ratio and al ferred to a solution of PTX (10 mg, 3.7 mmol) in anhydrous pyridine (2 lowed to react for l h at 4Â°C.Thereactive functionalities were quenched ml). The solution was stirred for 1.5 h at 23Â°C,and the solvents were by adding 2-mercaptoethylamine (0.1 ITIM)followed 1 min later by evaporated under reduced pressure. A solution of the residue in water (2 Â¿V-ethylmaleimide(0.2 RIM).Purification of the conjugate was achieved ml) was washed with CH^Cb (3 x 1 ml). The aqueous fraction was using Sepharose S-300 (Pharmacia) column and 10 RIMsodium phos lyophilized, and the product was purified on CM-Sephadex C-25 using phate, pH 7.2, containing 0.15 MNaCl as eluant. Fractions were mon 20 HIMsodium phosphate buffer, pH 4.5, as eluant. The yield of NH itored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, PAP was estimated to be 41% by UV spectroscopy. To remove trace absorbance at 280 nm, and for PGA activity using 6-nitro-3-(phenylac- amounts of PTX, the product was purified further on CM-Sephadex, etamido)benzoic acid (Sigma) as a substrate (35). Those fractions con resulting in a final yield of highly purified NHPAP of 10%. The purity sisting mainly of mAb:PGA 1:1 (by sodium dodecyl sulfate-polyacry of NHPAP was confirmed by UV spectroscopy, high pressure liquid chromatography, and 500 MHz 'H-NMR (dimethyl sulfoxide-i/6) lamide gel electrophoresis analysis) were combined and concentrated by ultrafiltration (Amicon PM-30 filter). The protein concentration was which showed new resonances at &10.21 (m), 7.92 (m), and 7.80 (m), determined spectrophotometrically at 280 nm using Â£Â°â€¢l%valuesof 1.4 corresponding to the amide protons, and an AB quartet at 5 7.02 (d, J = 8.1 Hz) and 6.65 (d, J = 8.1 Hz) corresponding to the new and 2.1 for L6 and PGA, respectively. Fluorescence activated cell sorter aromatic protons. NHPAP was stored at -20Â°C in 50% aqueous eth- analysis using a competitive binding assay (15) and enzyme activity measurements using 6-nitro-3-(phenylacetamido)benzoic acid as a sub anol. There was no apparent change in the NHPAP preparation by UV spectroscopy or in cytotoxic activity over a period of 6 months. strate (35) indicated that the properties of each of the proteins in the conjugate were largely unaffected by the manipulations described. Preparation of L6-PGA Cytotoxicity Assays To a 30Â°Csolution of PGA (5-10 mg/ml) in l ORINIsodium phos phate, pH 7.2, containing 0.15 MNaCL was added SMCC (Pierce; 20 The cytotoxic effects of PTX and NHPAP on cultured tumor cells RIMin dimethylformamide) so that the final concentration of SMCC were determined by measuring the incorporation of [l4C]leucine into was 0.6 RIM.After 30 min, the protein was separated from low molec protein, [3H]thymidJRe into DNA, or exclusion of trypan blue. ular weight materials on a Sephadex G-25 column that was eluted with Method A. H2981 cells were treated with trypsin/EDTA for 5 min at 37Â°C.The suspended cells were washed by centrifugaron with 40 RIMsodium phosphate containing 500 RIMNaCl at pH 7.2 and kept on ice prior to conjugation. The level of substitution was determined by LFMEM and resuspended to 2 x IO6 cells/ml in LFMEM with or reacting the maleimides with excess mercaptoethanol, followed by without PGA. EL4 or HSB-2 cells were harvested by centrifugation and quantification of the remaining thiol groups with 5,5'-dithiobis(2-ni- resuspended to 2 x 10" cells/ml in LFMEM Â±PGA. Cells treated with 5760

L6-PGA were incubated with the conjugate ( 10 Mg/ml)for 30-60 min at 100 r 4Â°Cinsterile polystyrene culture tubes prior to 4 washes with medium. Aliquots (50 ^1) of the cell suspension (IO5 cells) were added to 96-well plates, followed by the addition of PTX or NHPAP in 50 ^1 of 80 LFMEM. The cultures were incubated for a total of 18 h at 37Â°Cunder a humidified 7% CO2 atmosphere and then pulsed with [l4C]leucine (0.5 MCi/well in 50 n\ LFMEM) for 2 h. The cells were frozen, thawed, 5 60 and harvested onto glass-fiber filter disks. The filters were counted on n a Beckman LS 7500 scintillation counter. Method B. Cells in IMDM were plated into 96-well microtiter plates at 3000 cells/well and allowed to adhere overnight at 37Â°C.The IMDM 40 was removed, and various dilutions of NHPAP Â±PGA in 100 n\ of IMDM were added. Other drugs in IMDM were added to cells without PGA. After drug exposure at 37Â°C,the plates were washed, 100 n\ of 20 IMDM were added, and incubation was continued for approximately 24 h. The cells were pulsed with ['HJthymidine (1 Â¿iCi/wellin 100 n\ IMDM) for 24 h, frozen, thawed, and harvested onto glass-fiber filter disks. The filters were counted on an LKB /3-plate cell harvester. Cells that were treated with 5-fluorouracil were pulsed with ('H)leucine (1 Time (hours) MCi/well in 100 n\ IMDM) and compared to a similarly treated control. Fig. 3. Kinetics of cell kill by PTX. H298I cells were exposed to IO"8 MPTX, Method C. H2981 cells (IO7 cells/200 M!)were incubated at 4Â°Cin and cell viability was determined using a trypan blue exclusion assay according to method D in "Materials and Methods." IMDM (defined in method B) or in IMDM containing L6 mAb at 5 mg/ml. After 30 min, the cells were washed with IMDM (3x2 ml), and incubated at 4Â°Cfor 30 min in IMDM or in IMDM containing L6- PGA at 0.1 mg/ml. The cells were washed with IMDM (3x2 ml), treated with NHPAP or PTX in 200 ^1 IMDM, and incubated at 37Â°C NHPAP for 4 h. After a washing with IMDM (3 x 1 ml), the cells were diluted to IO5 cells/ml and plated out (100 ^I/well) in triplicate into 96-well plates. Following incubation at 37Â°Cfor24 h, the cells were pulsed with ['H]thymidine (1 ^Ci/well) for 24 h. The cells were detached using trypsin/EDTA and processed as in method B. Method D. H2981 cells in IMDM were distributed into polypropy lene tubes (8 x IO5cells/tube). The medium was removed, and the cells (at 37Â°C)weretreated with 1 ml of PTX ( 10 * Min medium) prewarmed to 37Â°C.Atvarious times, aliquots (20 M')were added to 80 ^1of trypan blue (Gibco; 0.4%) and counted using a hemocytometer. Concentration (M) Fig. 4. Cytotoxic effects of PTX and NHPAP Â±PGA (3.3 Â¿ig/ml)on EL4 murine T-cell lymphoma cells. Cells were exposed to the drugs for 18 h according RESULTS to method A in "Material and Methods." Points, mean of triplicate samples. Cytotoxicity of PTX and NHPAP. The cytotoxic effect of PTX on the H2981 human lung adenocarcinoma cell line was these experiments, H2981 cells were exposed to PTX at a con compared to a panel of clinically approved anticancer drugs. centration at least 1000 times greater than its IC50 value. Cells were exposed to the drugs for 1 h and washed, and via Within 20 min of PTX exposure, the H2981 cells took on a swollen appearance, and one-half of the cells lost their ability to bility was determined 24 h later by measuring the incorporation of [-1H]thymidine into DNA or [3H]leucine into protein. PTX exclude trypan blue by 1 h (Fig. 3). Thus, PTX is much more (IC.so, 3 x 10~12 M) was at least 5 orders of magnitude more toxic to cells than conventional chemotherapeutics and exerts toxic to H2981 cells than doxorubicin, mitomycin C, or 5-flu its activity rapidly. orouracil (Fig. 2). The preparation of NHPAP (Fig. 1) was achieved by reacting the primary amino group of PTX with an active ester of 4-hy- The kinetics of PTX mediated cell kill was determined using a trypan blue exclusion assay as a measure of cell viability. In droxyphenylacetic acid. NHPAP was purified from the reaction mixture by cation-exchange chromatography on two successive CM-Sephadex columns. High field 'H-NMR analysis of NH

100 PAP indicated the presence of aromatic and amide proton res onances which were not originally present in PTX. Both NH 80 PAP and PTX were hydrophilic and could be stored for I 5-FU prolonged periods (>6 months) at â€”¿20Â°Cwithoutany apparent PTX 60 o decomposition or loss of activity. o 40 A comparison of the cytotoxic effects of PTX and NHPAP r was made by incubating EL4 cells for 18 h with the drugs in the Doxorubicin 20 presence or absence of PGA at 3.3 Mg/nil. It was found that NHPAP was at least 1000 times less cytotoxic than PTX (Fig. ,o13 ,<512,0" iÃ³'Â°io9io8 io7 io6 io5 io4 io3 4). Incubation of the cells with a combination of NHPAP and PGA resulted in a level of cytotoxic activity that was equal to Concentration (M) that of PTX. PGA alone had no effect on cell viability. Similar Fig. 2. Cytoloxic effect of PTX on H2981 human lung adenocarcinoma cells experiments were performed on LX-1 (human lung adenocar compared to some clinically approved anticancer drugs. Cells were exposed to the drugs for I h. washed, and processed according to method B in "Materials and cinoma), H3639 (human ovarian carcinoma), and H3719 (hu Methods." Points, mean of triplicate samples; bars, SD. man colon carcinoma) cells (Table 1). The cytotoxic activity of 5761

Table 1 Cytotoxicity ofNHPAP Â±PGA on carcinoma cell lines (IC5() 4 x 10-" M) (Fig. 5B). These results demonstrate that (M)Â°NHPAP activation ofNHPAP occurs in an antigen-specific manner and CelllineLX-1 +PGA3 that the released drug is active in picomolar concentrations. x Kr9 x IO'12 A second method to establish that prodrug activation could H3639 4 X 10-' 3x IO-'2 be mediated through antigen bound enzyme conjugate involved 4 x 10-'IC50 7x IO-'2 H3719NHPAP3 an assay in which H2981 cells were saturated with mm iodi tied " Cells were exposed to varying concentrations of NHPAP Â±PGA (10 pg/ml for 4 h, washed, and processed according to method B in "Materials and Methods. L6 (5 mg/ml) prior to treatment with L6-PGA (0.1 mg/ml). After washing, the cells were exposed to NHPAP. The cyto toxic effect of this combination was compared to cells treated Table 2 Effect of drugs on parental and drug resistant cell lines with L6-PGA and no free L6 and to cells exposed only to (MC-AgentDoxorubicin IC5o NHPAP or PTX (Fig. 6). Again, PTX (IC50, 3 X IO"10 M)was much more cytotoxic than NHPAP (no cytotoxic effect up to 10~8 M).The reduced toxicities of PTX and NHPAP compared x IO"9 x IO"7 x IO'8 x 10~7 to Figs. 2,4, and 5 are most likely due to the high concentration Mitomycin C 7 x IO-7 1.5 x 10~6 9x 10-" 1 x 10~6 5-Fluorouracil 9x 10-' 3 x IO'4 5x 10-' 3 x 10-" of cells (5 x IO7 cells/ml) used in this experiment. NHPAP was NHPAP 3 x 10~8 3 x IO'8 5 x IO-8 5 x 10-* rendered significantly more cytotoxic (ICSO 3 x 10~9 M) on 4x 10-"HCT116(VM)46'114x 10-"A27801.52x 10-'Â°A2780DDP*12x 10-'Â° NHPAP + PGACHCT1167 H2981 cells that were treated with L6-PGA. When the binding " Cells were exposed to varying concentrations of drug for 4 h, washed, and processed according to method B in "Materials and Methods." of L6-PGA was inhibited with a large excess of unmodified L6, * Drug resistant cell line. prodrug activation was abrogated. This confirms that NHPAP c PGA concentration. 10 Mg/ml. can be activated by L6-PGA conjugate bound to H2981 target cells. NHPAP was increased approximately 1000-fold on all three cell lines by adding PGA to the culture medium. These results DISCUSSION are consistent with those obtained using EL4 cells (Fig. 4) and show that NHPAP is a prodrug that undergoes activation In order for the chemotherapeutic treatment of cancer to be through the catalytic activity of PGA. effective, the drugs that are used must be able to exploit subtle differences between neoplastic and normal tissues (36). Most The cell lines HCT116 (human colon carcinoma) and A2780 (human ovarian carcinoma) were also treated with PTX and clinically approved anticancer drugs exploit the increased NHPAP, and the cytotoxic effects were compared to those growth rate of tumor cells for therapeutic selectivity. Because obtained on the respective drug resistant cell lines, HCT116- PTX binds with high affinity to cell surface receptors present on (VM)46 (33) and A2780DDP (34). Within each pair of cell both cycling and quiescent mammalian cells, it is unlikely that systemically administered PTX will act selectively upon tumor lines, no differences in sensitivities toward NHPAP or the com cells (25-27). However, intratumoral generation of PTX from bination of NHPAP with PGA were observed (Table 2). This was in contrast to doxorubicin, mitomycin C, and 5-fluorou- less toxic derivatives may direct its biodistribution and allow for racil, which proved to be significantly more cytotoxic to the its use in the clinic. parental cell lines compared to the corresponding drug resistant PTX displays an extraordinary level of cytotoxic activity on a variety of cell lines (Tables 1, 2) as evidenced by the fact that it lines (Table 2). is 5-8 orders of magnitude more potent than some commonly Conjugate Preparation. An immunoconjugate of PGA was prepared with the mAb L6 (31, 32) through a stable thioether bond. This was accomplished by adding 1-2 maleimide groups to PGA with the heterobifunctional cross-linking reagent, SMCC, and reducing 1-2 of the native disulfides in L6 with dithiothreitol. The proteins were combined, allowed to react, and purified by gel filtration. The conjugate consisted primarily of material having a L6:PGA ratio of 1:1. The combined mo lecular weight of these proteins is 250,000. A competition bind ing assay (15) using fluorescence activated cell sorter analysis indicated that the ability of the mAb moiety of the immuno conjugate to bind to antigens on the H2981 carcinoma was preserved. Assays of enzyme activity were performed using 6-nitro-3-(phenylacetamido)benzoic acid as a substrate (35). There was no loss in enzymatic activity as a result of the reac tions or other manipulations required for conjugate prepara tion. Yields generally ranged between 20 and 30%. Antigen Directed Cytotoxicity. The H2981 human lung adenocarcinoma (L6 antigen positive) and HSB-2 human T-lymphoma (L6 antigen negative) cell lines were used to determine 10 the ability of an L6-PGA conjugate to activate NHPAP. Both cell lines were equally sensitive to PTX (IC50 10~n M) and to Concentration (M) Fig. 5. Immunologically specific activation ofNHPAP. H2981 cells (A) (L6 NHPAP (IC50 10~8 M)(Fig. 5, A and B). However, upon treat antigen positive) and HSB-2 cells (B) (L6 antigen negative) were treated with ment with L6-PGA, the H2981 antigen positive cells were L6-PGA (10 Mg/ml). washed, treated with NHPAP for 18 h. and processed ac much more sensitive to NHPAP (IC50 6 x IO"" M) (Fig. 5/0 cording to method A in "Material and Methods." This was compared to the effects of PTX and NHPAP on previously untreated cells. Points, mean of trip compared to similarly treated HSB-2 antigen negative cells licate samples. 5762

been shown to possess increased levels of mÃ¤rI mRNA and NHPAP P-glycoprotein (33). The multidrug resistance of A2780DDP ~i|_6-H-6-PGA has been attributed to increased intracellular pools of folates + NHPAP and thymidylate synthase (34). HCT116(VM)46 was shown to be resistant to doxorubicin (14-fold), mitomycin C (2-fold), and 5-fluorouracil (3-fold) compared to the parental cell line. The A2780DDP cell line also displayed resistance to doxorubicin (7-fold), mitomycin C (11-fold), and 5-fiuorouracil (6-fold). Significantly, the drug resistant and respective parental cell lines responded identically to treatment with NHPAP Â±PGA. The mechanisms responsible for the drug resistance of these cells lines evidently afford no protection against the product of Concentration (M) NHPAP hydrolysis. In conclusion, a relatively nontoxic derivative of PTX can be Fig. 6. Effect of inhibiting conjugate binding. H2981 cells were treated with L6 mAb at saturating levels (5 mg/ml). washed, treated with L6-PGA conjugate at activated on cell surfaces by mAb-enzyme conjugates. The drug saturating levels (O.I mg/ml), and then exposed to varying concentrations of NHPAP according to method C in "Materials and Methods." The cytotoxic that is released acts extracellularly and has unusually high po effects were compared to cells that did not receive L6 or L6-PGA before exposure tency, a rapid cytotoxic effect, and the potential of circumvent to NHPAP or PTX and to cells that were treated only with L6-PGA (0.1 mg/ml) ing some of the more common forms of multidrug resistance. In before exposure to NHPAP. Points, mean of triplicate samples. vivo experiments are currently being performed.

used anticancer drugs (Fig. 2). Cell death occurs rapidly (50% ACKNOWLEDGMENTS cell kill in 1 h) after exposure to PTX (Fig. 3). Indeed, PTX exhibits a level of potency far greater than all other drugs that We are grateful to Carolyn Lazo and Melissa Churley for their ex have been generated from prodrug/mAb-enzyme combinations cellent technical assistance and to Dr. Bala S. Krishnan for discussions (6-19). and spectral data. Elucidation of some of the structural requirements for PTX activity was reported by Wattenberg et al. (26), who prepared REFERENCES the jV-acetyl and /V-(p-bromobenzoyl) derivative of the toxin. 1. Eichler, H. G. Clinical experience of targeted therapy. Biotherapy, 3: 77-85, They proposed that since these derivatives were approximately 1991. 100-fold less toxic to Swiss 3T3 cells than PTX, the amino 2. Wawrzynczak, E. J. Systemic immunotoxin therapy of cancer: advances and prospects. Br. J. Cancer, 64: 624-630, 1991. terminus is an important structural feature that may affect the 3. Pimm, M. V. Drug-monoclonal antibody conjugates for cancer therapy: po affinity of PTX for its receptor. Consistent with this is our tential and limitations. CRC Crii. Rev. Ther. Drug Carrier Syst., 5:189-227, finding that NHPAP is significantly less toxic (approximately 1988. 4. Vitella. E. S., Fulton. R. J., May. R. D., Till, M., and Uhr, J. W. Redesigning 1000-fold) than PTX on several cell lines (Table 1; Fig. 4). It is nature's poisons to create anti-tumor reagents. Science (Washington DC), unclear whether the residual activity of NHPAP is due to in 238: 1098-1104. 1987. herent toxicity of the prodrug, low levels of hydrolysis under the 5. Thorpe, P. E. Antibody carriers of cytotoxic agents in cancer therapy. A review. In: A. Pinchera. G. Doria, F. Dammacco, and A. Bargellesi (cds.). culture conditions, or to traces of contaminating PTX that were Monoclonal Antibodies '84: Biological and Clinical Applications, pp. 475- below the detection limit of NMR and high pressure liquid 506, Milan: Editrice Kurtis. 1985. 6. Scnter, P. D. Activation of prodrugs by antibody-enzyme conjugates: a new chromatography. If the latter condition applies, the amount of approach to cancer therapy. FASEB J., 4: 188-193. 1990. free PTX in the prodrug preparation must be less than 0.1%, 7. Senter, P. D., Saulnier. M. G., Schreiber, G. J., Hirschberg, D. L., Brown, J. based on the differences in cytotoxic activities between PTX P., HellstrÃ¶m, I., and HellstrÃ¶m, K. E. Anti-tumor effects of antibody-alka line phosphatase conjugates in combination with etoposide phosphate. Proc. and NHPAP. Nati. Acad. Sci. USA, 85: 4842-4846, 1988. The activation of NHPAP by conjugates of L6-PGA was 8. HellstrÃ¶m, K. E., and Senter, P. D. Activation of prodrugs by targeted en shown by two independent methods to take place in an immu- zymes. Eur. J. Cancer, 27: 1342-1343, 1991. nologically specific manner (Figs. 5 and 6). L6-PGA that was 9. 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. bound to cell surface antigens on H2981 cells effected a 100- Ablation of human choriocarcinoma xenografts in nude mice by antibody- fold increase in NHPAP cytotoxicity. The fact that the level of directed enzyme prodrug therapy (ADEPT) with three novel compounds. Eur. J. Cancer, 27: 1361-1366, 1991. activation did not quite equal the activity of PTX might be due 10. Bagshawe, K. D., Springer, C. J., Searle, F., Antoniw, P.. Sharma, S. K., to incomplete or slow enzymatic conversion of NHPAP to Melton, R. G., and Sherwood, R. F. A cytotoxic agent can be generated PTX. 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Gary S. Bignami, Peter D. Senter, Paul G. Grothaus, et al.

Cancer Res 1992;52:5759-5764.

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