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Bioconjugate Chem. 2003, 14, 302−310
Synthesis and Biological Evaluation of Paclitaxel-C225 Conjugate as a Model for Targeted Drug Delivery1 Ahmad Safavy,*,†,| James A. Bonner,†,| Harlan W. Waksal,⊥ Donald J. Buchsbaum,† G. Yancey Gillespie,‡ M.B. Khazaeli,† Ramin Arani,§ Dung-Tsa Chen,§ Mark Carpenter,§ and Kevin P. Raisch† Departments of Radiation Oncology and Surgery, Biostatistics Unit, and Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, Alabama 35294, and ImClone Systems, Somerville, New Jersey 08876. Received April 24, 2002; Revised Manuscript Received October 1, 2002
Tumor-targeted drug delivery is an attractive strategy in cancer treatment. We have previously reported a paclitaxel model conjugate using a bombesin receptor-recognizing peptide in which the drug cytotoxicity against H1299 human nonsmall cell lung cancer was enhanced compared to unconjugated taxol. In an effort to expand the development of tumor-recognizing taxanes, paclitaxel (PTX, taxol) was conjugated to the anti-epidermal growth factor receptor (anti-EGFR) monoclonal antibody (MAb) Erbitux (C225) to serve as a model MAb-mediated drug delivery compound. Thus, paclitaxel was derivatized at its 2′-hydroxy function by introduction of a succinate linker, and the carboxyl group of the latter was covalently attached to C225 through amide bond formation. The final product conjugate (PTXC225) was analyzed mass spectrometrically for assessment of the drugto-antibody ratios. Cytotoxicity screening of the drug-antibody conjugate against A431, UM-SCC-1, and UM-SCC-6 cells indicated an enhancement in cytocidal effect of paclitaxel as compared to those of the free drug, the intact antibody, and a physical mixture of the two (the controls). In A431 cells, the conjugate showed 25.2% ( 2.2% of apoptosis induction as compared to little or no apoptosis caused by the controls. Biodistribution analysis of the PTXC225 in tumor-implanted nude mice and a tyrosinekinase assay showed that conjugation of the drug did not interfere with the immunoreactivity of the antibody. The 24-h tumor uptake of C225 and PTXC225 were 11.7% ( 6.0% and 7.1% ( 3.6% of the injected dose per gram of tissue (%ID/g), respectively, which were not significantly different. Also, in A431-implanted nude mice, the conjugate and C225 showed tumor growth inhibition effects of 57.2% and 41.2%, respectively, against a saline-treated control, which were not significantly different from each other. This lack of difference in the in vivo antitumor activity of the MAb-delivered drug and free PTX may be due to either a relatively low dose of the antibody-delivered drug (346 µg/kg), or an untimely release of it, or both. The tumor growth inhibition pattern of the conjugate, however, was identical to that of C225, indicating that the attachment of PTX did not affect the antigen-binding and growth inhibitory features of the MAb. These preliminary results demonstrate the potential of tumor-targeted delivery of taxol as a promising strategy in cancer treatment and warrant further work to develop more suitable drug-MAb linkers as well as improved dosage and treatment protocols.
INTRODUCTION
Although a powerful and important modality in cancer treatment, the effectiveness of chemotherapy is limited by the efficiency of the delivery of the drug to the tumor site(s), systemic toxicity, and side-effects of the agents used. The delicate balance between the toxicity and sideeffects prevention, and producing the maximum therapeutic efficacy, is usually hard to achieve and requires long and tedious protocol development procedures which may jeopardize patient survival and quality of life. Development of antitumor drug delivery systems capable of tipping this balance in favor of a positive outcome of the therapy without worsening the side-effects would be highly desirable. * Corresponding author: Ahmad Safavy, Ph.D.,1824 6th Avenue South, WTI 674, Birmingham, AL 35294-6832. Telephone: (205) 934-7077; Fax: (205) 975-7060; e-mail:
[email protected]. † Department of Radiation Oncology. ‡ Department of Surgery. § Biostatistics Unit. | Comprehensive Cancer Center. ⊥ ImClone Systems.
A potential strategy in achieving this goal may be drug delivery through a tumor-specific mechanism (1-3). Targeted treatment of neoplastic disease has advanced considerably in the last two decades, after the establishment of monoclonal antibody (MAb)2 technology by Kohler and Milstein (4). An early realization of this technology was in the development of radiolabeled Mabs, which have reached clinical use for imaging and therapy 1 Presented in part at the Eighth Conference on Radioimmunodetection and Radioimmunotherapy of Cancer, Princeton, New Jersey, 12-14 October, 2000. 2 Abbreviations: BSA, bovine serum albumin; DMEM, Dulbecco’s modified Eagle medium; DMF, N,N-dimethylformamide; DPBS, Dulbecco’s phosphate-buffered saline; EEDQ, 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline; EGFR, epidermal growth factor receptor; FBS, fetal bovine serum; HSR, hypersensitivity reaction; ip, intraperitoneal or intraperitoneally; iv, intravenous or intravenously; MAb, monoclonal antibody; MALDI-TOF, matrix-assisted laser desorption/ionization-time-of-flight; MS, mass spectrometry; MW, molecular weight; NHS, N-hydroxysuccinimide; PTX, paclitaxel; PTXSX, PTX-hemisuccinate; sc, subcutaneous or subcutaneously; SDM, standard deviation of the mean; TCC, targeted cancer chemotherapy.
10.1021/bc020033z CCC: $25.00 © 2003 American Chemical Society Published on Web 01/28/2003
Paclitaxel−Monoclonal Antibody for Targeted Therapy
(5, 6), with some still under study for future clinical applications (7). Drug-MAb conjugates have also been studied in cancer therapy, albeit to a lesser extent, and the field is still open to much improvement (8, 9). Conjugates of the widely used anticancer drug doxorubicin have been reported (8-12) in attempts to both ameliorate the significant toxicities of this drug and to enhance its efficacy. Hamann and co-workers have reported conjugates of the antitumor antibiotic calicheamicin with the anti-CD33 MAb P67.6 for treatment of acute myeloid leukemia (13, 14). After our initial report on the synthesis and in vitro cytotoxicity evaluation of the first taxol-MAb conjugate (15), Correa and Page´ (16), and Guillemard and Saragovi (17), reported preparation and cell growth inhibition studies of PTX-BCM43/2E5 conjugate, and PTX-MC192 and PTX-5C3 conjugates, respectively. The latter report also included an in vivo tumor growth inhibition experiment for one conjugate (PTX-MC192). With the exception of two time-points and only in one cell line, the IC50 values of Correa and Page´ were close to or higher than those of free paclitaxel. In the other report, enhancement in cytotoxicity of roughly 26-33% against B104 rat neuroblastoma cells, with respect to free paclitaxel, were reported with only 9% improvement in in vivo tumor inhibition in a nude mice model bearing sc B104 xenografts (17). The interest to develop tumor-targeting drug-MAb conjugates stems from a duality in character of most antitumor agents, that is, the ability to eradicate cancer cells and toxicity to normal tissue. Despite their potential and an impressive clinical record, taxane drugs also suffer from dose-limiting toxicities. An added drawback of the native taxol is its extremely low aqueous solubility, a mere 0.25 mg/mL. This has led to the currently used Taxol formulation (Bristol-Myers Squibb, Princeton, NJ) of this drug consisting of 30 mg of PTX in 5 mL of a 50/50 mixture of Cremophore EL (polyethoxylated castor oil, a solubilizing surfactant) and dehydrated ethanol, which yields a homogeneous parenteral preparation. Likewise, the clinical formulation of docetaxel (Taxotere, Rhone-Poulenc Rorer, Collegeville, PA) uses polysorbate 80 and 13% ethanol in water. Both formulations have been reported to cause medium to severe hypersensitivity reactions (HSRs). Cremophore EL has been reported to cause histamine release resulting in severe allergic reactions (18, 19). In fact, the early clinical trials of taxol were delayed due to the HSRs until an effective premedication regimen was developed (20). Because of these problems, the National Cancer Institute recommended a 24-h infusion protocol for taxol before which time the patient was premedicated with antiallergy drugs. In addition to HSRs, some major side effects are associated with the use of taxanes (21-25). A peripheral neuropathy is induced by PTX, which becomes more severe by cumulative dosing, and an arthralgia-myalgia syndrome resulting 2-5 days after administration of taxol which is more pronounced with shorter infusion schedules (26). Other systemic toxicities include cardiac arrhythmias, alopecia, mucositis, and fatigue (20). Skin and nail toxicities such as onycholysis, dermatitis, and reactive erythema (27, 28) and cardiac conduction disturbances (29) are among other side effects resulting from the toxicities of this drug. Research on the design and development of water-soluble and tumor-specific taxane drugs, which could be formulated without the use of allergenic excipients and possess higher therapeutic indices without increasing the effective dose, is therefore well justified.
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We have reported the first PTXMAb and PTX-peptide conjugates designed for tumor-targeted delivery of this agent and have demonstrated the enhanced cytotoxic properties of the drug in controlled in vitro experiments (15, 30, 31). Here, we report accounts of synthesis, cytotoxicity, apoptosis induction, biodistribution, and a preliminary antitumor activity evaluation of a PTXMAb conjugate using the human-mouse chimeric anti-EGFR antibody C225 (32, 33). EXPERIMENTAL SECTION
General Procedure for the Synthesis of PTXMAb Conjugates. Paclitaxel (Hande Tech, Houston, TX) was derivatized according to the procedure of Deutsch et al., by succinic anhydride in pyridine, to paclitaxel-2′-succinate (PTXSX) (34). The carboxyl function of the PTXSX (2 mg, 2.1 µmol) was activated by the addition of NHS (0.27 mg, 2.3 µmol) and EEDQ (0.52 mg, 2.1 µmol) in dry DMF (250 µL) and at 0 °C for 1 h. The DMF solution of the activated ester (15.5 µL, containing 0.13 µmol of PTXNHS) was then added to a precooled (0 °C) solution of C225 (2 mg, 0.013 µmol) in 600 µL of PBS (25 mM, pH 8.1). The solution was stirred at this temperature for 1 h at which time the reaction progress was confirmed by MALDI-TOF MS and using BSA as a MW calibrator. The conjugate was purified by dialysis in DPBS (4 × 2 L) at a MW cutoff of 50 kDa. The protein content of all samples were measured by the method of Lowry (35). The PTXC225 conjugates used in this work had a drug-toMAb molar ratio of 1.6 or 2 by MALDI. In the following in vitro and in vivo experiments, the molar concentrations of the unconjugated PTX and C225 controls were adjusted according to these ratios as indicated. Tumor Cell Lines. A431 human epidermoid cancer cells were obtained from the American Type Culture Collection (Manassas, VA). The A431 cell line was maintained in Dulbecco’s modified Eagle’s medium (DMEM): F12 (50:50) containing 7% FBS. The head and neck squamous cell carcinoma lines (UM-SCC-1 and UM-SCC6) were maintained in DMEM containing 10% FBS. All cell lines were supplemented with L-glutamine, penicillin, and streptomycin and incubated at 37 °C in 5% CO2. The UM-SCC-1 and UM-SCC-6 lines were provided by Dr. Thomas Carey (University of Michigan). Assessment of EGFR Recognition and Receptor Phosphorylation. A431 cells were plated in six-well plates at a density of 100 000 cells/well and allowed to grow for 48 h. The cells were treated with 1 µg/mL each of C225 or PTXC225 conjugate for 24 h, after which time they were exposed to 10 nM EGF (Sigma Chemical Co., St. Louis, MO) for 5 min. Cell lysates were collected in lysis buffer (0.025 M Tris‚HCl, pH 7.5, 0.25 M NaCl, 0.005 M EDTA, 1% (v/v) NP-40, 0.001 M phenylmethylsulfonyl fluoride, 2 µg/mL aprotinin, 2 µg/mL leupeptin, 0.001 M sodium orthovanadate, 0.05 M sodium fluoride and 0.03 M sodium pyrophosphate, as described previously (36). Equal amounts of protein (5 µg) were loaded per lane and separated by 10% SDS-PAGE and transferred to Immobilon-P membrane (Millipore Corp., Bedford, MA). The immunoblots were blocked in 10% milk/Trisbuffered saline with Tween-20 (TBS-T) (20 mM Tris‚HCl, pH 7.5, 137 mM NaCl, 0.05% Tween-20) for 1 h at room temperature. The membrane was incubated with the primary antibody, anti-EGFR (Sigma Chemical Co.), or anti-phosphotyrosine (Santa Cruz Biotechnology Inc, Santa Cruz, CA) in 3% milk/TBS-T overnight at 4 °C.
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The secondary anti-mouse IgG-HRP antibody (Sigma Chemical Co.) was incubated with the membrane at room temperature for 1 h. The blots were developed by chemiluminescence (Amersham Life Sciences Inc, Arlington Heights, IL). In Vitro Cell Proliferation Assay. The cells were plated in 24-well plates at a density of 10 000 cells/well and allowed to grow for 48 h, at which time (day 0) the cells were treated with C225 (3.2 nM), PTX (6.4 nM), C225 (3.2 nM) + PTX (6.4 nM), or PTXC225 conjugate (3.2 nM for a PTX:MAb of 2) for 24 h. The treatments were removed on day 1 and were replaced with drugfree medium. The cells were counted on day 4 and normalized to the percent of untreated cells. Each treatment was done in quadruplicate. Apoptosis-Induction Assay. An Annexin V-FITC Apoptosis Detection kit (BioVision, Inc., Palo Alto, CA) was used to examine the induction of apoptosis. A431 cells were plated at a density of 40 000 cells per well in six-well plates. After 24 h, fresh culture medium was added to the appropriate wells containing the following agents: C225 (3.2 nM), PTX (6.4 nM), C225 (3.2 nM) + taxol (6.4 nM), or PTXC225 conjugate (3.2 nM for a PTX: C225 of 2). At 24 h posttreatment, the agents were removed and replaced with drug-free medium. The cells were collected 72 h later according to the manufacturer’s protocol. Briefly, the trypsinized cells were pooled with the corresponding culture medium containing nonadherent cells. The cells were separated from the medium by centrifugation at 300g for 5 min. The cell pellets were suspended in 500 µL ice-cold 1× binding buffer. Annexin V FITC and propidium iodide were added to the cell suspension followed by a 10 min incubation at room temperature in the dark. The cells were collected by flow cytometry and analyzed by CellQuest v3.1 software (Bectin Dickinson, San Jose, CA), and results are presented as percent average cell count ( SD. All treatments were done in triplicate. 125I-Labeling. A standard iodogen radiolabeling method was used (37). Briefly, 10 mg of iodogen was dissolved in 1 mL of chloroform and 5 mL of this solution was dispensed into a vial containing a polystyrene bead. The bead was then dried under an argon flow. Sodium [125I]-iodide (5 mCi/bead) was added, followed by 5× (v/v) of a pH 6 phosphate buffer. The C225 or PTXC225 was added, and the mixture was incubated for 15 min. The labeled MAb was purified on a PD-10 column (Pharmacia, Uppsala, Sweden) eluted with 0.1% HSA in DPBS. Animal Model. Athymic nude female nu/nu mice with a BALB/c background were obtained from the National Cancer Institute Frederick Research Laboratory (Frederick, MD) and were kept under sterile conditions. Procedures to minimize discomfort, pain, and distress were in accord with the Animal Resource Program at the University of Alabama at Birmingham, accredited by the American Association for Accreditation of Laboratory Animal Care. The A431 cells were harvested and suspended in sterile PBS at a concentration of 7.5 × 108 viable cells/mL. Cell viability was determined by trypan blue dye exclusion. Viable cells (107) in sterile PBS were injected sc into the right flank. Biodistribution of C225 and PTXC225. Groups of mice (six per group) with established 7-day sc tumors received 5 µCi of the labeled antibody or conjugate as an iv bolus injection. At 4 and 24 h postinjection, the animals were exsanguinated after anesthesia and dissected. Samples of blood, heart, lung, liver, stomach, small intestine, spleen, kidney, skin, bone, muscle, uterus,
Safavy et al.
Figure 1. Schematic representation of a PTXMAb conjugate in which the linkage is through the 2′-hydroxy group of the drug. Scheme 1
pancreas, and tumor were blotted dry, weighed, and counted for the uptake of radioactivity in a well-type gamma counter (Minaxi-gamma 5000 series, Packard, Chicago, IL). Results of biodistribution were expressed as mean ( SD % ID/g (percent injected dose per gram of tissue) at 4 and 24 h postinjection. In Vivo Tumor Growth Inhibition Study. Athymic nude female mice (n ) 6, nu/nu, 19-22 g body weight) were implanted sc with A431 cells (5 × 106) on the right flank. At day 10, tumors were measured by vernier calipers, and tumor volumes were calculated and mice were sorted into three groups, evenly distributing tumors by volume size. The animals were injected ip with saline, C225 (894 µg), and the PTXC225 (894 µg) conjugate with a PTX:MAb of 1.6 (8.05 µg PTX). Tumor sizes were monitored and increases in fractional tumor volumes (FTVs) were calculated from the following equation:
FTV ) Voldn/Vold1 where Voldn and Vold1 are volumes at days n and 1, respectively. Statistical Methods. The %ID/g values were compared between the group with injection of C225 antibody and the group injected with the PTXC225. Group difference may indicate there exists interaction of C225 and PTX. Two sample t tests were used to detect the difference (38). If both groups have substantially different percentage of dose/gram, the p value produced by the t test will be small (
