Generation of an Intensely Potent Anthracycline by a Monoclonal

Seattle Genetics, 21823 30th Drive SE, Bothell, Washington 98021, and University of Texas M.D. Anderson. Cancer Center, Houston, TX, 77025. Received ...
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Bioconjugate Chem. 2005, 16, 717−721

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Generation of an Intensely Potent Anthracycline by a Monoclonal Antibody-β-Galactosidase Conjugate Michael Y. Torgov,† Stephen C. Alley,† Charles G. Cerveny,† David Farquhar,‡ and Peter D. Senter†,* Seattle Genetics, 21823 30th Drive SE, Bothell, Washington 98021, and University of Texas M.D. Anderson Cancer Center, Houston, TX, 77025. Received February 14, 2005; Revised Manuscript Received April 1, 2005

The L49 monoclonal antibody against the p97 antigen on melanomas and carcinomas was chemically conjugated to E. coli β-galactosidase (β-gal), forming a largely monomeric conjugate with preserved enzymatic activity. The resulting L49-β-gal conjugate was used to activate (N-[(4′′R,S)-4′′-hexyloxy4′′-(1′′′-O-β-D-galactopyranosyl)butyl]daunorubicin) (1), a derivative of daunorubicin that has low cytotoxicity and high chemical stability. Addition of the conjugate to the prodrug resulted in an increase in cytotoxicity of approximately 105-fold, a level of activation that is higher than any mAb-enzyme/ prodrug combination yet described. Furthermore, the released drug had an IC50 value of approximately 10 pM, making it significantly more potent than the vast majority of clinically approved anticancer drugs. The potential of this enzyme/prodrug combination for cancer therapy is discussed.

INTRODUCTION

A considerable amount of research has surrounded the use of monoclonal antibody (mAb)-enzyme conjugates for the activation of anticancer prodrugs (1, 2). This approach to cancer chemotherapy, referred to as ADEPT (antibodydirected enzyme prodrug therapy) involves two separate steps to achieve therapeutic efficacy. Initially, a mAbenzyme conjugate is administered, which localizes within the tumor mass and clears from the systemic circulation over time. In the second step an anticancer prodrug is administered, which circulates throughout the body and is converted to active drug by the mAb-enzyme conjugate. Depending on the tumor to nontumor conjugate ratio, the prodrug can be very well-tolerated, and high intratumoral drug concentrations are obtained (1-4). The strength of this approach is evident from not only the large number of mAb-enzyme/prodrug combinations that have preclinical activity, but also from the indications of efficacy in early-stage clinical trials (1, 5). One of the interesting aspects of ADEPT is that it allows not only for intratumoral generation of clinically approved drugs such as doxorubicin (6), paclitaxel (7), etoposide (8), 5-fluorouracil (4), and melphalan (9) but also of drugs that would be too toxic for use in a nontargeted form. Such drugs include potent minor groove binders (10), bis-iodomustards (11), cyanide (12), and the marine natural product palytoxin (13). The rationale for generating highly potent drugs is that the amount of localized enzyme within solid tumors is likely to be very low (14), and this will restrict the number of drug molecules that are generated. Therefore, maximal therapeutic efficacy is likely to be obtained through the intratumoral generation of highly potent cytotoxic agents. ADEPT systems that lead to such drugs would not only require a high tumor to nontumor conjugate ratio, but also a prodrug with greatly attenuated toxicity. In this paper, we describe a mAb-β-galactosidase (βgal1) conjugate for the activation of an intensely potent * To whom inquiries should be directed at [email protected]. † Seattle Genetics. ‡ University of Texas M.D. Anderson Cancer Center.

daunorubicin derivative. Previous studies have shown that treatment of a galactosyl derivative of daunorubicin (1, Figure 1A) with unconjugated β-gal released a daunorubicin derivative that was 100-1000 times more potent than unmodified daunorubicin (15, 16). This enzyme has also been used to release such agents as CC-1065 derivatives (10, 17) and daunorubicin (18). The only mAbconjugated form of β-gal used for prodrug activation involved a derivative of 5-fluorouridine (19). Here, we extend these findings by demonstrating that 1 can be activated by as much as 105-fold by a mAb-β-gal conjugate that binds to the p97 antigen on melanomas and some carcinomas. This represents one of the highest cytotoxic differentials between a prodrug and potent drug for application in ADEPT. EXPERIMENTAL PROCEDURES

General Analytical Methods and Materials. The murine L49 mAb was obtained as previously described (20). E. coli β-gal was purchased from Calbiochem (San Diego, CA). N-(-maleimidocaproic acid)hydrazide (EMCH), DTNB, and the BCA protein assay reagent were from Pierce (Rockford, IL). (N-[(4′′R,S)-4′′-Hexyloxy-4′′-(1′′′-Oβ-D-galactopyranosyl)butyl]daunorubicin) (1) and (N-(5,5diacetoxypent-1-yl)doxorubicin) (5) were prepared as previously described (15, 16). Protein concentrations of unmodified L49 and β-gal were determined by UV absorption spectrometry at 280 nm, and those of conjugated enzyme were determined using the BCA protein assay reagent. All measurements were made against known concentrations of β-gal as a reference standard. Oxidation of L49 and Conjugation to β-Gal. NaIO4 (final concentration 12.5 mM) was added to L49 in 50 mM sodium acetate (pH 5.5) and 50 µM methionine. The reaction was allowed to proceed in the dark for 50 min at ambient temperature. The oxidized L49 was desalted and buffer exchanged into PBS using a PD-10 column (Amersham Biosciences, Piscataway, NJ). EMCH was 1 Abbreviations used: EMCH, N-(-maleimidocaproic acid)hydrazide; β-gal, β-galactosidase; 4-MUG, 4-methylumbelliferylβ-D-galactopyranoside.

10.1021/bc050039z CCC: $30.25 © 2005 American Chemical Society Published on Web 04/27/2005

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Figure 1. A. Mechanism of activation of 1 by β-gal. B. Formation of the analogous iminium ion 6 by 5.

then added at a 50-fold molar excess over L49 and allowed to react for 4 h at ambient temperature with 10% DMSO present to ensure the solubility of EMCH. L49EMCH was then purified using a PD-10 column preequilibrated with PBS. The number of the introduced maleimide residues was determined by treating with 50 µM cysteine and quantitating the remaining unreacted cysteine with DTNB. β-Gal was added to L49-EMCH at 0.5:1 enzyme to antibody molar ratio, and the reaction was allowed to incubate overnight at 4 °C. The conjugate was purified from unreacted L49 by SEC HPLC on a TSKgel G3000SW column (Tosoh Bioscience, Montgomeryville, PA) using PBS containing 10 mM MgSO4 as the mobile phase. After concentration, the L49-β-gal conjugate was stored at 4 °C. Amino Acid Analysis for the Determination of Conjugation Ratio. Amino acid analysis was performed on the L49-β-gal conjugate by the molecular structure facility at the University of California at Davis. The experimental mole fraction of each amino acid in the conjugate (MFAAA) was determined from these data. The theoretical mole fraction of each amino acid was calculated from the primary amino acid sequence of L49 (MFL49) and β-gal (MFβ-gal). The stoichiometry of the L49-β-gal conjugate (X moles of L49 per 1 mole of β-gal) is related to the experimental and theoretical mole fraction by eq 1:

MFAAA )

XmMFL49 + 1nMFβ-gal Xm + 1n

(1)

where m and n are the number of occurrences of the amino acid in L49 and β-gal, respectively. Solving for X for each amino acid yielded an average value of 0.7 mol of L49 per mole of β-gal.

Enzyme Activity Assays. β-Gal activity of the conjugate was measured using 4-methylumbelliferyl-β-Dgalactopyranoside (4-MUG) or 1 as substrates. Dilutions of the enzyme or the conjugate (1 µg per data point) in 25 µL of PBS with 10 mM MgSO4 were incubated for 5 min at 37 °C and then mixed with an equal volume of the substrate in the same buffer. The final substrate concentration varied from 80 to 8 µM. During a 20 min incubation course at 37 °C, 50 µL aliquots were taken and quenched with an equal volume of methanol. Aliquots (100 uL) were analyzed by HPLC on a 4.6 × 150 mm, 4u Synergi MAX-RP 80 column (Phenomenex, Torrance, CA) using the following gradient at 1 mL min-1: solvent A 0.1% trifluoroacetic acid in H2O; solvent B 0.1% trifluoroacetic acid in acetonitrile; a 10-min linear gradient from 10% A to 90% A. Peak areas at 495 or 315 nm for 1 and 4-MUG, respectively, were used to calculate concentrations of the product based on the peak area at time infinity. Eady-Hofstee plots of velocity versus the ratio of velocity and substrate concentration were used to calculate Vmax and Km. In Vitro Cytotoxicity Assay. H3677 melanoma cell line seeded at a density of 2 × 103 per well in a 96-well plate and allowed to adhere overnight in RPMI 1640 medium containing 10% FBS in the absence of antibiotics. Serial dilutions of the L49-β-gal conjugate and an L49 control were added, incubated for 1 h at room temperature, and then washed. The prodrugs 1 or 5 were then added for 24 h. Cultures were washed and grown for an additional 92 h, at which point Alamar Blue (Biosource International, Camarillo, CA) was added to 10% of the total culture volume. Cells were incubated for 4 h, and dye reduction was measured on a Fusion HT fluorescent plate reader (Packard Instruments, Meriden, CT).

mAb−β-Galactosidase Conjugate

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Figure 2. Mechanism of conjugate formation. L49 oxidized with sodium periodate (L49-CHO) reacts with EMCH to form L49EMCH. The endogenous thiols of β-gal (β-gal-SH) then reacts with the maleimide to form the L49-β-gal conjugate. RESULTS AND DISCUSSION

Prodrug Design. The structure of the known daunorubicin analogue 1 (16) is shown in Figure 1. This previously reported compound has been shown to give rise to an intensely potent metabolite in the presence of β-gal. In phosphate buffer at 37 °C, 1 is slowly degraded with a half-life of 49 h (16, 21). The proposed mechanism of action is that hemiacetal 2, formed by the cleavage of the glycosyl bond of 1, spontaneously loses hexanol to form aldehyde 3, which then spontaneously cyclizes leading to the generation of iminium ion 4 (Figure 1A). Figure 1B shows the mechanism of formation of 6, a compound related in structure to 4, from N-(5,5-diacetoxypent-1-yl)doxorubicin (5) (15). Thus, compound 5, which was designed to be hydrolyzed by cellular esterases, can be used to gauge the potential differences in cytotoxic activities between 1 and the unstable hydrolysis product 4. DNA interstrand cross-links in HL60 human leukemia cells have been shown and proposed for compound 5 and 1, respectively (15, 22). Thus, these compounds differ mechanistically from the topoisomerase II inhibitor daunorubicin. L49-β-Gal Conjugate Preparation. E. coli-derived β-gal is a tetrameric enzyme composed of 116 kDa monomers that has previously been used successfully for anticancer prodrug activation (19, 23). The enzyme has a neutral pH optimum while mammalian β-gal has optimal activity under acidic conditions (24, 25). Bacterial and mammalian β-gal activities have been successfully distinguished in transgenic mice by carefully controlling pH (26). Because of these differences in pH optima, it was anticipated that the prodrugs would undergo minimal nonspecific activation when administered in vivo. To prepare the L49-β-gal conjugate, the carbohydrates of L49 were oxidized with sodium periodate, generating aldehydes on the heavy chain in the vicinity of the hinge region. The aldehydes were subsequently reacted with N-(-maleimidocaproic acid)hydrazide (EMCH) to yield a maleimide-activated antibody. These were coupled with β-gal, which contains endogenous sulfhydryl groups (Figure 2). L49 was added in a 2-fold excess with respect to the enzyme to minimize the amount of unreacted enzyme remaining after the conjugation reaction. The resulting conjugate, whose formation was manifested by the forward shift relative to the β-gal peak, was purified from unreacted L49 by size-exclusion HPLC (Figure 3A). SDS-PAGE analysis further confirmed the presence of the cross-linked material (Figure 3B). β-gal is a tetramer composed of 116 kDa monomers. The conjugation conditions were targeted toward 1:1 cross-linking ratio, e.g. a single antibody molecule per enzyme tetramer. Therefore, under both reducing and nonreducing conditions, a dominant 116 kDa β-gal band is present that corresponds to the non-cross-linked subunit in the enzyme tetramer. A cross-linked product represented by two bands of approximately 160 kDa is consistent with

Figure 3. A. Size-exclusion chromatography HPLC traces for L49-β-gal conjugate (solid line), β-gal (dotted line), and L49 (dashed line) using PBS with 10 mM MgSO4 as a mobile phase. B. SDS-PAGE (4-12%) of L49, β-gal, and L49-β-gal conjugate under reducing (with DTT) and nonreducing (without DTT) conditions. The arrowheads represent a contaminant present in the enzyme preparation. Lane 1, L49 with DTT; lane 2, β-gal with DTT; lane 3, L49-β-gal conjugate with DTT; lane 4, L49 without DTT; lane 5, β-gal without DTT; lane 6, L49-β-gal conjugate without DTT; and lane 7, molecular weight markers. C. SDS-PAGE (4-12%) of authentic mixtures of β-gal and L49 compared to L49-β-gal conjugate. All lanes are under reducing conditions. The arrowheads represent a contaminant present in the enzyme preparation. Lane 1, 1:0.5 mixture of β-gal and L49; lane 2, 1:1 mixture of β-gal and L49; lane 3, L49-β-gal conjugate; lane 4, L49; lane 5, β-gal; lane 6, molecular weight markers.

the expected combined size of a single enzyme subunit and a L49 heavy chain. In addition, under reducing conditions both heavy and light antibody chains are present in the lane with conjugate. However, their relative intensities are skewed toward the light chain due to sequestration of the heavy chain into conjugate (compare lane 1 and 3). Amino acid analysis performed to assess the antibodyto-enzyme ratio revealed that the conjugate is composed of 0.7 mole of L49 per 1 mole of enzyme. When L49 and β-gal were mixed at different molar ratios and compared by SDS PAGE to the conjugate, it was confirmed that the composition of the conjugate lies between 0.5 and 1 molecule of L49 per molecule of the enzyme tetramer, consistent with the amino acid analysis data (Figure 3C).

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Table 1. Enzyme Kinetic Parameters of L49 and L49-β-Gala β-gal activity compound

substrate

Km (µM)

Vmax (µM‚min-1‚mg-1)

β-gal

1 4-MUG 1 4-MUG

38 ( 22 22 60 ( 16 21.4

0.052 ( 0.025 89.6 0.035 ( 0.01 78.4

L49-β-gal

a Values shown are the average of a minimum of two independent experiments, except for 4-MUG (performed once).

Enzyme Activity. To test the enzyme activity, either 1 or 4-methylumbelliferyl-β-D-galactopyranoside (4-MUG) was added at concentrations ranging from 8 to 80 µM to β-gal or the L49-β-gal conjugate. Aliquots were precipitated with methanol and the substrate conversion was monitored by reversed-phase HPLC. The results, summarized in Table 1, demonstrate that the Km and Vmax values on both substrates were similar for the conjugate and free enzyme. This indicates that the conjugation chemistry did not impair the enzymatic activity of β-gal in the L49-β-gal conjugate. It should be noted that the specific activity values are in good agreement with those reported for a related molecule, gal-DNC4 (21). The slow rate of drug release most likely involves hydrolysis of the glycosidic bond as the rate limiting step, as significantly improved release kinetics were obtained using 4-MUG, which incorporates a better leaving group. In Vitro Studies. The cytotoxic activities of L49-βgal in combination with 1 were determined using the p97 positive H3677 human melanoma cell line. Cells were preincubated with the conjugate for 1 h, washed to remove the unbound material, and then exposed to 1 for a total of 24 h. Following the drug removal, the cytotoxic effects relative to untreated cells were determined using Alamar Blue as an indicator of cell viability. N-(5,5Diacetoxypent-1-yl)doxorubicin (5) was used as a control in the assay since it generates the active doxorubicin iminium ion 6, analogous to the daunorubicin iminium ion 4 generated from 1. The difference in cytotoxicity between 5 and 1 was approximately 105-fold. Consistent with published data (15), 5 had an IC50 value of 20 pM (Figure 4A). In contrast, the IC50 of prodrug 1 alone was 0.8 µM. Preincubation of cells with L49 in the presence of prodrug 1 did not have any effect on the cytotoxicity of 1. However, when the L49-β-gal conjugate was used, 1 exhibited cytotoxicity similar to 5. These data are consistent with the conclusions that 4 is generated as a result of galactoside cleavage, and that the galactoside of 1 is completely cleaved by β-gal. Next, we sought to establish the minimal concentration of the conjugate required to attain complete prodrug activation. Serial dilutions of the conjugate were incubated with the cells as described above, and any unbound conjugate was removed by washing. The prodrug 1 was then added at a constant concentration of 10 nM, significantly lower than its IC50 value. The assay demonstrated that a conjugate concentration of 40 nM (6 µg/ mL) led to 50% cell kill (Figure 4B). Thus, the two assays show that efficient prodrug activation is achieved and that the difference in prodrug and drug toxicity is pronounced. CONCLUSIONS

We have demonstrated that a chemical conjugate of L49-β-gal activates 1, with a cytotoxic differential of approximately 5 orders of magnitude. This degree of prodrug activation has not been demonstrated previously

Figure 4. In vitro cytotoxicity of 1 and 5 on H3677 cells detected by Alamar Blue reduction. Cells were treated with L49 or L49-β-gal conjugate for 1 h, washed, and then treated with 1 or 5. A. Cells were treated with 100 nM of L49 or L49-β-gal conjugate and the concentration of 1 or 5 varied. Open squares, 5; open triangles, 1; closed triangles, L49 plus 1; closed squares, L49-β-gal plus 1. B. Cells were treated with varied concentration of L49 or L49-β-gal conjugate and then 10 nM 1. Open squares, 1 plus L49; closed squares, 1 plus L49-β-gal.

in other ADEPT systems, which typically provide differences in the range of 10 to 100-fold (1, 2). The results reported here strongly suggest that attachment of the β-gal residue to daunorubicin leads to almost complete drug inactivation, and that the glycosidic bond is highly stable under the conditions used in the in vitro assay. These are necessary requirements in developing an ADEPT system involving a highly potent drug component. An additional feature of this system that distinguishes it from many other ADEPT systems is that the released drug is capable of circumventing the MDR phenotype (15). This latter property is critical, since it is highly likely that most patients who receive mAbenzyme/prodrug combinations will have already been treated extensively with conventional chemotherapy. While drug potency has not yet been shown to be a critical parameter for therapeutic efficacy in ADEPT, it may well be if, as expected, very small percentages of the injected conjugate accumulate within solid tumor masses. The results reported here justify future studies surrounding the in vivo activities of L49-β-gal/galactosyldaunorubicin combinations. It is expected that optimization will require the development of an engineered fusion protein containing an L49 fragment, since such

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molecules have demonstrated much higher tumor to normal tissue ratios than chemically derived conjugates (20, 27). In addition, forms of β-gal that are minimally immunogenic will be of greatest clinical utility. The development of molecules such as these will be the subject of future investigations. LITERATURE CITED (1) Bagshawe, K. D., Sharma, S. K., and Begent, R. H. (2004) Antibody-directed enzyme prodrug therapy (ADEPT) for cancer. Expert Opin. Biol. Ther. 4, 1777-89. (2) Senter, P. D., and Springer, C. J. (2001) Selective activation of anticancer prodrugs by monoclonal antibody-enzyme conjugates. Adv. Drug Delivery Rev. 53, 247-64. (3) Bosslet, K., Czech, J., and Hoffmann, D. (1994) Tumorselective prodrug activation by fusion protein-mediated catalysis. Cancer Res. 54, 2151-9. (4) Wallace, P. M., MacMaster, J. F., Smith, V. F., Kerr, D. E., Senter, P. D., and Cosand, W. L. (1994) Intratumoral generation of 5-fluorouracil mediated by an antibody-cytosine deaminase conjugate in combination with 5-fluorocytosine. Cancer Res. 54, 2719-23. (5) Francis, R. J., Sharma, S. K., Springer, C., Green, A. J., Hope-Stone, L. D., Sena, L., Martin, J., Adamson, K. L., Robbins, A., Gumbrell, L., O’Malley, D., Tsiompanou, E., Shahbakhti, H., Webley, S., Hochhauser, D., Hilson, A. J., Blakey, D., and Begent, R. H. (2002) A phase I trial of antibody directed enzyme prodrug therapy (ADEPT) in patients with advanced colorectal carcinoma or other CEA producing tumours. Br. J. Cancer 87, 600-7. (6) HariKrishna, D., Rao, A. R., and Krishna, D. R. (2003) Selective activation of anthracycline prodrugs for use in conjunction with ADEPT. Drug News Perspect. 16, 309-18. (7) Rodrigues, M. L., Carter, P., Wirth, C., Mullins, S., Lee, A., and Blackburn, B. K. (1995) Synthesis and beta-lactamasemediated activation of a cephalosporin-taxol prodrug. Chem. Biol. 2, 223-7. (8) 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, 4842-6. (9) Vrudhula, V. M., Kerr, D. E., Siemers, N. O., Dubowchik, G. M., and Senter, P. D. (2003) Cephalosporin prodrugs of paclitaxel for immunologically specific activation by L-49-sFvbeta-lactamase fusion protein. Bioorg. Med. Chem. Lett. 13, 539-42. (10) Tietze, L. F., Herzig, T., Fecher, A., Haunert, F., and Schuberth, I. (2001) Highly selective glycosylated prodrugs of cytostatic CC-1065 analogues for antibody-directed enzyme tumor therapy. Chembiochem 2, 758-65. (11) Niculescu-Duvaz, D., Niculescu-Duvaz, I., Friedlos, F., Martin, J., Lehouritis, P., Marais, R., and Springer, C. J. (2003) Self-immolative nitrogen mustards prodrugs cleavable by carboxypeptidase G2 (CPG2) showing large cytotoxicity differentials in GDEPT. J. Med. Chem. 46, 1690-705. (12) Kousparou, C. A., Epenetos, A. A., and Deonarain, M. P. (2002) Antibody-guided enzyme therapy of cancer producing cyanide results in necrosis of targeted cells. Int. J. Cancer 99, 138-48. (13) Bignami, G. S., Senter, P. D., Grothaus, P. G., Fischer, K. J., Humphreys, T., and Wallace, P. M. (1992) N-(4′-hydroxyphenylacetyl)palytoxin: a palytoxin prodrug that can be activated by a monoclonal antibody-penicillin G amidase conjugate. Cancer Res. 52, 5759-64.

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