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This maleimido derivative of geldanamycin (GMB-. APA-GA) was linked to the monoclonal antibody Herceptin after the antibody had been modified with...
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Bioconjugate Chem. 2002, 13, 786−791

Modifications in Synthesis Strategy Improve the Yield and Efficacy of Geldanamycin-Herceptin Immunoconjugates Raya Mandler,† Hisataka Kobayashi,† Marie Y. Davis,† Thomas A. Waldmann,† and Martin W. Brechbiel*,‡ Metabolism Branch and the Chemistry Section, Radiation Oncology Branch, National Cancer Institute, NIH, Bethesda, Maryland 20982-1002. Received December 26, 2001; Revised Manuscript Received April 15, 2002

Geldanamycin (GA) was modified with N-tert-butyloxycarbonyl-1,3-diaminopropane to introduce a latent primary amine. After deprotection, this primary amine provided a site for introduction of a maleimide group that enabled linkage to proteins. This maleimido derivative of geldanamycin (GMBAPA-GA) was linked to the monoclonal antibody Herceptin after the antibody had been modified with Traut’s reagent to introduce thiol groups. By this sequence, a new immunoconjugate (H:APA-GA) was generated that showed greater antiproliferative activity than the previously reported analogous immunoconjugate created with a 1,4-diaminobutane spacer derivative of geldanamycin to form an immunoconjugate, H:ABA-GA. Both immunoconjugates inhibited in vitro the growth of MDA-361/ DYT2 cells, a cell line overexpressing the HER2 antigen, while Herceptin alone was ineffective. However, H:APA-GA showed better efficacy than H:ABA-GA (IC50 ) 0.2 vs 0.58 mg/mL and cell doubling time >12 vs 6 days, respectively). Results of the in vivo therapy experiments in a xenograft model were consistent with the in vitro findings. Treatment with Herceptin prolonged the survival of the tumor-bearing mice when compared with the control group, but H:ABA-GA and H:APA-GA were each more efficacious than unmodified Herceptin. However, unlike H:ABA-GA, the immunoconjugate H:APA-GA caused stable tumor regression (in 25% of the recipients), showing a qualitative improvement with potential clinical relevance.

INTRODUCTION

Geldanamycin (GA)1 (Figure 1) is a benzoquinoid ansamycin produced by the actinomycete Streptomyces hygroscopicus that is related to herbimycin A with a known cellular target being the cytosolic protein chaperone hsp90 (1). Upon binding to hsp90, GA inhibits its ability to protect cellular enzymes from proteasomal degradation and disrupting in this way many crucial cellular processes (2). Consequently, this molecule is extremely cytotoxic to a wide variety of cells, including neoplastic cells. While the antitumor potential of GA has long been recognized, clinical use of native GA has not been pursued due to the drug’s severe toxicity and difficulties with aqueous formulation (3). Numerous GA analogues have been synthesized and studied in an effort to develop derivatives with improved therapeutic indices (4). These studies indicated that modifications at the 17 position on the quinone ring maintained anticancer activity at nanomolar range while reducing the overall toxicity (3, 4). One of these derivatives, 17-allylamino* To whom correspondence should be addressed: NIH, Chemistry Section, 10 Center Drive, Building 10, Room B3B69, Bethesda, MD 20892-1002. E-mail: [email protected]. † Metabolism Branch, National Cancer Institute. ‡ Chemistry Section, National Cancer Institute. 1 Abbreviations: BOC, tert-butyuloxycarbonyl; GA, geldanamycin; mAb, monoclonal antibody; H:ABA-GA, Herceptin conjugated with 17-(3-[4-maleimidobutyrcarboxamido]butylamino)geldanamycin; H:APA-GA, Herceptin conjugated with 17-(3-[4maleimidobutyrcarboxamido]propylamino)geldanamycin; GMB, 4-maleimidobutyric acid N-hydroxysuccinimidyl ester; GMBABA-GA, 17-(3-[4-maleimidobutyrcarboxamido]butylamino)geldanamycin; GMB-ABA-GA, 17-(3-[4-maleimidobutyrcarboxamido]propylamino)geldanamycin.

GA (Figure 1), is currently entering Phase II clinical trials to treat a variety of solid tumors (5, 6). Further reduction of GA systemic toxicity could potentially be achieved by selectively targeting and delivering an active GA species into malignant cells using a monoclonal antibody (mAb) as the targeting vehicle. Clearly, internalization is a prerequisite for successful application of such immunoconjugates (7). With the advances of therapeutic mAbs into clinical use, it is conceivable that mAbs that internalize efficiently could also be used as the targeting vehicle in such an application (8). Several tumor-targeting mAb’s armed with small toxic compounds have already been clinically evaluated and one has been approved for therapy of AML in elderly patients (9, 10). In our present studies, Herceptin was chosen as the targeting mAb. This mAb was the first to be approved by the FDA for therapy of solid tumors (11). Herceptin specifically targets the membrane receptor HER2, a member of the epidermal growth factor receptor family. HER2 is overexpressed in approximately 30% of human gastric, lung, and breast carcinomas and is associated with poor prognosis. Blocking of HER2 activity or interfering with its expression was shown to inhibit proliferation and reduce tumor growth (11-13). Because HER2 stability and function depends on hsp90, this protein is highly sensitive to and is efficiently eliminated by GA (14). Herceptin has been previously shown to internalize upon binding to HER2. The conjugation with GA is through an acid-labile bond that should release the GA derivative once the endocytosed conjugate is shuttled into the lysosomes. In fact, our laboratories have already reported that such conjugates do deliver a more potent selective cytotoxic impact than use of just Herceptin (1517).

10.1021/bc010124g Not subject to U.S. Copyright. Published 2002 by American Chemical Society Published on Web 06/19/2002

Modifications in Synthesis of Immunoconjugates

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Figure 1. Structures of geldanamycin, 17-GMB-ABA-GA, 17allylamino-GA.

Conjugation of mAb’s with GA has not been a straightforward process because GA lacks a suitable functional group for direct covalent linkage to protein. As recently reported by our laboratories, introduction of a primary amine group originating from modification at the 17 position has been successful and permitted subsequent addition of a heterobifunctional cross-linking reagent for conjugation of the modified GA to mAb’s (15, 16). By use of this chemistry, 17-GMB-ABA-GA (Figure 1) could be conjugated to Herceptin efficiently and reproducibly. The product immunoconjugate, H:ABA-GA, subsequently demonstrated a higher antiproliferative effect on HER2 overexpressing cells than unmodified Herceptin. H:ABAGA was also shown to be specific for HER2-positive, but not for HER2-negative, cells. The choice of 17-GMB-ABAGA rather than the more potent 17-GMB-APA-GA, butyl spacer versus propyl spacer, respectively, was the result of a compromise between activity and availability of the required GA derivative (15, 16). While 17-APA-GA had higher antiproliferative activity than 17-ABA-GA, the preparation of 17-GMB-APA-GA has been problematic. Simple introduction of the amine via direct aminolysis with 1,3-diaminopropane yielded a complex mixture of byproducts (15). Exposure to silica during chromatography aggravated this condition, efficiently catalyzing the formation of a seven-membered imine ring from the distal amine and the quinone carbonyl, further complicating access to this derivative. Insertion of an additional methylene into the amine chain eliminated this cyclization (15) and thus provided 17-GMB-ABA-GA in excellent yield. This synthesis ultimately allowed for the synthesis of a very pure and active GA-mAb immunoconjugate. (15)

Figure 2. Synthesis of 17-GMB-APA-GA.

However, this strategy compromised the potency of the final immunoconjugate, prompting re-examination of the chemical strategies that had been employed. Herein, we report on a successful modified synthesis strategy of 17-GMB-APA-GA (Figure 2), its conjugation to Herceptin, and an evaluation of this immunoconjugate’s efficacy in vitro as well as in tumor-bearing mice. EXPERIMENTAL PROCEDURES

Materials and Methods. All anhydrous solvents and reagents except where noted otherwise were obtained from Aldrich, Sigma, or Fluka and were used as received. The N-hydroxysuccinimidyl ester of 4-maleimidobutyric acid (GMB) was prepared by modification of a literature method (18). Geldanamycin (GA) was generously supplied by Edward A. Sausville and Gordon Cragg, NPG, DTP, DCTD, NCI. Triethylamine was distilled from CaH2 prior to use. 1 H and 13C NMR were obtained using a Varian Gemini 300 instrument, and chemical shifts are reported in ppm

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on the δ scale relative to TMS, TSP, or solvent. Proton chemical shifts are annotated as follows: ppm (multiplicity, integral, coupling constant (Hz)). Chemical ionization mass spectra (CI-MS) were obtained on a Finnegan 3000 instrument. Fast atom bombardment mass spectra (FABMS) were obtained on an Extrel 4000 in the positive ion detection mode. Elemental analyses were performed by Atlantic Microlabs (Norcross, GA). The MDA-361/DYT2 cell line was generously provided by Drs. Dajun Yang and Marc E. Lippman of The Lombardi Cancer Center, Georgetown University (Washington, DC) (19). The NIH3T3/HER cell line was generously provided by Dr. S. Aaronson, NIH (20). 125I was obtained in the NaI form from Amersham Biosciences Corp. (Piscataway, NJ). Caution: 125I (t1/2 ) 59.4 day) is a γ-emitting radionuclide. Appropriate shielding and handling protocols should be in place when using this isotope. All protocols in which animals were used have been approved by the National Cancer Institute Animal Care and Use Committee. 17-(3-tert-Butyloxycarbonyl(aminopropyl)amino)geldanamycin (17-N-BOC-APA-GA). GA (0.5 g, 0.89 mmol) was dissolved in CHCl3 (300 mL), and N-tertbutyloxcarbonyl-1,3-diaminopropane (250 mg, 1.44 mmol) was added. The reaction was stirred shielded from light for 48 h, during which the initial golden yellow solution grew darker and an additional 100 mg (0.575 mmol) of the monoprotected diamine was added. After 96 h, the reaction was assayed by thin-layer chromatography (TLC) on silica (5% MeOH/CHCl3). Despite the now dark purple appearance of the reaction, a distinct yellow component corresponding to GA was still evident by TLC, and another 100 mg of the protected diamine was added. After 120 h, the amount of GA remaining was deemed minimal by TLC, the reaction solution was concentrated to ca. 25 mL, and hexanes (ca. 450 mL) was added to precipitate the crude product. The suspension was placed at -20 °C for 24 h, after which the dark purple solid was collected on a Buchner funnel, washed with hexanes, and dried in vacuo in the absence of light. The pure 17-NBOC-APA-GA was cleanly isolated by flash chromatography on silica (2.5 × 35 cm column) eluting with 1:99 MeOH/CHCl3 (470 mg, 75%). 1H NMR (300 MHz, CDCl3, δ): 0.991 (br, d, 6H, J ) 5.7 Hz), 1.432 (m, 1H), 1.456 (s, 9H), 1.803 (s, 3H), 1.835 (m, 2H), 2.028 (s, 3H), 2.410 (dd, 1H, J ) 12.9, 10.8 Hz), 2.61-2.82 (m, 3H), 3.239 (m, 1H), 3.276 (s, 3H), 3.366 (s, 3H), 3.40-3.70 (m, 6H), 4.309 (d, 1H, J ) 9.9 Hz), 4.693 (t, 2H, J ) 6.3 Hz), 4.837 (br, s, 2H), 5.185 (s, 1H), 5.81-5.95 (m, 2H), 6.586 (t, 2H, J ) 11.7 Hz, one proton obscured), 6.955 (d, 1H, J ) 11.7 Hz), 7.267 (s, 1H), 9.167 (s, 1H). FAB-MS (nba/CsI) m/e: 704 (M+). Anal. Calcd for C36H56N4O10: C, 61.33; H, 8.02; N, 7.95. Found: C, 61.45; H, 8.37; N, 7.72. 17-(3-(4-Maleimidobutyrcarboxamido)propylamino)geldanamycin (17-GMB-APA-GA). The 17-NBOC-APA-GA (100 mg, 0.142 mmol) was dissolved in CH2Cl2 (100 mL) and treated with trifluoroacetic acid (10 mL) at room temperature for 1 h, after which TLC indicated that starting material was no longer present. The solution was rotary-evaporated at ambient temperature until near dry. The residue was taken up in CH2Cl2 (ca. 50 mL) and rotary-evaporated until near dry 3 times and then finally dried in vacuo for 24 h. The crude protonated amine was taken up in ethyl acetate (100 mL), and GMB (80 mg, 0.286 mmol) was added to the solution. Immediately thereafter, triethylamine (100 µL) was added, and the reaction was stirred in the dark for 24 h. At this point, TLC on silica with 10% MeOH/CHCl3

Mandler et al.

indicated one major component, and the solvent was removed by rotary evaporation at ambient temperature. The product was isolated by flash chromatography on silica as described previously, eluting with 2% MeOH/ CHCl3 (90 mg, 83%). 1H NMR (300 MHz, CDCl3, δ): 0.990 (t, 6H, J ) 7.5 Hz), 1.77-1.60 (m, 3H), 1.802 (s, 3H), 1.99-1.84 (m, 3H), 2.029 (s, 3H), 2.184 (t, 2H, J ) 7.2 Hz), 2.44-2.32 (m, 2H), 2.61-2.80 (m, 3H), 3.275 (s, 3H), 3.48-3.38 (m, 2H), 3.367 (s, 3H), 3.38-3.66 (m, 6H), 4.314 (d, 1H, J ) 9.9 Hz), 5.191 (s, 1H), 5.81-5.95 (m, 2H), 6.044 (t, 1H, J ) 5.7 Hz), 6.588 (t, 1H, J ) 11.4 Hz), 6.626 (t, 1H, J ) 5.7 Hz), 6.726 (s, 2H), 6.956 (d, 1H, J ) 12.0 Hz), 7.263 (s, 1H), 9.164 (s, 1H). FAB-MS (nba/prg/ CsI) m/e: 767 (M+). HR-FAB-MS m/e: 767.3742, error ) -0.7 ppm. Anal. Calcd for C39H53N5O11: C, 60.99; H, 6.97; N, 9.12. Found: C, 61.18; H, 7.04; N, 8.97. Conjugation of GA Derivatives (17-GMB-ABA-GA, 17-GMB-APA-GA) to Herceptin (H:ABA-GA, H:APAGA). The conjugation was performed as previously reported (15, 16). Herceptin was reacted with Traut’s reagent at a molar ratio of 1:13. Excess Traut’s reagent was removed by buffer exchange into conjugation buffer (50 mM HEPES, 150 mM NaCl, 10 mM EDTA (pH 7.0)). The molarity of the SH functionality groups after thiolation was determined by the method of the Ellman reaction at A412 (16). When compared with the molarity of the mAb itself, this value allowed calculation of the average reaction ratio of GA:mAb. The reaction conditions employed yielded an average of three thiol groups per protein molecule. Thereafter, 17-GMB-APA-GA (or 17-GMB-ABA-GA) was dissolved in DMSO (2 mg/mL) and was reacted with the mAb at g5-fold the concentration of the mAb’s SH groups. The reaction mixture was kept in the dark at 25 °C for 1 h and then was dialyzed extensively (3 × 1 L during 48 h) against PBS without Ca2+/Mg2+ at 4 °C. The presence of the GA moiety on the mAb was confirmed by spectrophotometric reading at A334, as previously reported (15, 16). All conjugates were filtered for sterility and were kept at 15-20 mg/mL, at 4 °C. Humanized anti-CD25 mAb (Zenapax, HoffmanLaRoche) was similarly treated to produce a comparable immunoconjugate as previously reported (16). Immunoreactivity and Cell Binding Assay. To establish that the conjugation did not compromise the mAb, antigen recognition and cell binding of the immunoconjugates were compared to native Herceptin. MDA-361/ DYT2 cells as well as the HER2-transfectant cell line NIH3T3/HER were used as a source of HER2 for these assays. The transfectant cells were employed because they expressed a much higher receptor/cell number than MDA-361/DYT2 cells (2.3 × 106 vs 0.18 × 106 receptor/ cell, respectively, as determined by Scatchard plot analysis) and thus provided conditions for a more accurate evaluation of antigen binding. Herceptin and H:APA-GA were each labeled using 125I (7 mCi/mg of protein) employing a modified Chloramine-T method. Immunoreactivity was determined by a modified cell-binding assay (21, 22). Aliquots of the 125I-labeled H:APA-GA or parental Herceptin (2 ng/100 µL) were incubated in parallel with 5.0 × 105 to 2.0 × 106 NIH3T3/HER cells or 1.0 × 106 MDA-361/DYT2 cells for 2 h at 4 °C. Nonspecific binding was determined under conditions of excess antibody (25 µg of nonradiolabeled Herceptin). The Kd values for Herceptin and the GA immunoconjugate were obtained from the Scatchard plot analysis. Evaluation of In Vitro Efficacy of H:APA-GA. MDA-361/DYT2 cells were seeded into six-well, flatbottom plates at 5 × 104 cells/mL and allowed to adhere. Herceptin and the two GA immunoconjugates were each

Modifications in Synthesis of Immunoconjugates

added to the wells at 0.5 mg/mL (2 mL). Control treatments were PBS only (vehicle) and 1 µM BOC-APA-GA (2 mL). To calculate cell doubling times, cells were detached and counted every 48 h, for 12 days. To establish an IC50 for the conjugates, MDA-361/DYT2 cells were seeded into 96-well plates and were treated with increasing concentrations of Herceptin and either of the two conjugates, as described previously (15, 16). Cultures were allowed to grow until the PBS-treated controls reached 80% confluency. Cell growth was measured after fixing the cultures in all wells, staining the fixed cells with Crystal Violet, and reading the eluted dye at A540 (15). Evaluation of Therapeutic Efficacy of the H:APAGA with Human Xenografts in Athymic Mice. Sixweek old athymic female mice (NCI, Frederick, MD) were inoculated sc with MDA-361/DYT2 cells (2 × 106/0.1 mL). These tumors are estrogen-dependent; therefore, E2 pellets (Innovative Research of America, Sarasota, FL) were inserted under the skin 24 h prior to tumor transplanation (0.72 β-estradiol, constant release for 60 days). When tumors reached 15-20 mm3, the mice were randomly divided into groups (n ) 8) and treatment was initiated. The treatment regimen was an ip dose of the immunoconjugate at 4 mg/Kg in PBS (0.2 mL) every 3 days for 120 days. The size of the tumors and their appearance was monitored and recorded during that time. Mice were terminated when the tumors reached ∼1200 mm3, if the tumor became necrotic, or if the animals showed signs of severe stress and illness. RESULTS AND DISCUSSION

The synthesis of the 17-GMB-APA-GA (Figure 2) was performed using the mono-BOC-protected 1,3-diaminopropane. This completely eliminated the competing formation of the seven-membered ring that occurred with use of just the diamine. Thus, treatment of GA with N-tert-butyloxcarbonyl-1,3-diaminopropane (Figure 2) produced a dark purple solid that was isolated by precipitation with hexane and then easily purified by column chromatography on silica gel. The most effective reaction conditions involved a careful titration of the GA with the protected amine over several days while carefully following the progress of the reaction by TLC. Regardless of this care, isolation of a minor product that appeared to have added two equivalents of protected diamine (as determined by mass spectrometry) was still observed. This product was presumed to form from the addition of a second equivalent of the monoprotected diamine to the quinone carbonyl, forming an imine. The rationale for using the tert-butylcarbamate (BOC) protecting group was that the formed primary amine would be efficiently protonated upon acidic removal of the BOC group and would effectively continue to inhibit the competing cyclization reaction. Although acidic deprotonation and interception of the amine was expected to lead to the desired product, this was not altogether assured. Deprotection with trifluoroacetic acid appeared to be extremely efficient. However, initial attempts to first directly generate the free amine and subsequently perform reactions with the N-hydroxysuccinic ester of γ-maleimidobutyric acid (GMB) led to capricious results. In contrast, reproducible results were only obtained by generating the free amine in situ as the final component of this reaction, concomitantly trapping the amine with the GMB. As such, routine production of the GA derivative, 17-GMB-ABA-GA, suitable for conjugation to proteins or peptides in reasonably large amounts was achieved in good yield and excellent purity.

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Figure 3. Effect of GA immunoconjugates on cellular growth rate of HER2-overexpressing cells. MDA-361/DYT2 cells were treated with 0.5 mg/mL Herceptin or GA immunoconjugate. Cell numbers were determined every 48 h, for 12 days. Cell doubling times for the PBS, Herceptin, H:ABA-GA, and H:APA-GAtreated cultures were 1.4, 1.6, 6.2, and greater than 12 days, respectively.

This product, as well as the previously reported 17GMB-ABA-GA, were each conjugated to Herceptin, as described in the Experimental Procedures (15, 16). Previously, the purity of the 17-GMB-ABA-GA had been assessed by analytical SE-HPLC and shown to be essentially congruent with Herceptin (15). This observation was duplicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Thus, in this current study, SDS-PAGE was employed to confirm the absences of conjugation reaction byproducts. All of the conjugates employed herein were found to be comparable to Herceptin as determined by SDS-PAGE (data not shown). The immunoreactivity of H:APA-GA was not compromised by this conjugation process to any detectable level as measured in binding assays. The absolute amount of bound immunoconjugate to MDA-361/DYT2 cells was 13% ( 1% for both H:APA-GA and parental Herceptin using 1.0 × 106 cells. Binding was even higher (∼91 ( 2% for Herceptin and H:APA-GA) when the assay was performed with NIH3T3/HER cells, consistent with the fact that the amount of HER2 antigen was higher on the surface of these cells. To support the characterization of the immunoconjugate, the Kd values for Herceptin and the H:APA-GA, as obtained from Scatchard plot analysis, were determined. The Kd values clearly were not significantly different, 4.2 ×and 3.8 × 10-9 M for H:APA-GA and Herceptin, respectively, and were consistent with previously reported values for Herceptin (23). The biological activity of 17-APA-GA was reported to be approximately 3-fold higher than that of 17-ABA-GA (16). Thus, the immunoconjugate formed with 17-GMBAPA-GA, H:APA-GA, was expected to exert a stronger inhibitory activity compared with H:ABA-GA. The antiproliferative activity of the two immunoconjugates was evaluated using MDA-361/DYT2 cells and is displayed in Figure 3. Cell growth rate was measured in cultures treated with either 0.5 mg/mL of Herceptin, H:ABA-GA, or H:APA-GA. The effect of Herceptin alone was marginal

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Figure 5. Effect of GA immunoconjugates on survival of MDA361/DYT2 sc xenograft (15-20 mm3) bearing athymic mice (n ) 8/group). Reagents were given ip at 3 day intervals at 4 mg/ kg and were continued for 3 months.

Figure 4. Dose-response curves obtained from MDA-361/ DYT2 cultures treated with increasing concentrations of either Herceptin, GA immunoconjugates, or GA derivatives alone. Doses are expressed in molarity of GA moeity, other than in the Herceptin alone samples. All samples were run in duplicate and are presented with SD bars.

while both immunoconjugates inhibited cellular growth. As expected, the H:APA-GA showed higher efficacy than H:ABA-GA, maintaining the cultures at no apparent growth for 8 days. Cell doubling times for the control (PBS) and Herceptin-treated cultures were 1.4 and 1.6 days, respectively, while for H:ABA-GA this was 6.2 days. In contrast, the doubling time of the H:APA-GA-treated cultures exceeded 12 days. The IC50 values of H:APAGA and H:ABA-GA were determined from seven separate experiments to be 0.2 ( 0.03 and 0.58 ( 0.02 mg/mL, respectively, and a representative experiment is shown in Figure 4. In contrast, IC50 values for native Herceptin could not be determined because the maximal inhibition of unmodified Herceptin reached only 25%, even at concentrations higher than 10 mg/mL (Figure 4). The specificity of cell targeting was addressed in our earlier publications on GA immunoconjugates (15-17). In brief, anti-HER2:GA immunoconjugates were demonstrated to not inhibit proliferation of HER2-negative cells, such as the lymphocytic cell line HuT102. These immunoconjugates also did not inhibit the proliferation carcinoma cells with normal HER2 expression, such as A431 or MCF-7 cells. The inverse condition was also demonstrated, that is, that GA immunoconjugates comprised of irrelevant mAbs did not affect HER2-positive cells and that, in all cases, selective internalization was crucial for efficacy. Concurrent with our ongoing studies, Kasuya et al. reported a similar finding concerning the isolable nature of the propylamine-derivatized GA via a protonation procedure that was used postreaction between GA and 1,3-diaminopropane (24). The procedure provided here is believed to offer some advantages in that complete obviation of the competing cyclization reaction was achieved and that both the carbamate-protected GA and protonated-deprotected GA are then readily available for subsequent chemical modification. While chromatographic separation steps were required to obtain the carbamate-protected GA as well as the 17-GMB-APA-GA in high purity, these operations were not difficult. In the case of the former compound, this did eliminate the

consistently present impurity that arose from addition of a second molecule of reagent to GA. Recently, Kasuya et al. also reported the preparation of a GA immunoconjugate that targets ovarian cancer cells (25). They employed different chemistry creating a polymeric form of APA-GA with N-(2-hydroxyproply)methacrylamide (HPMA) that could be conjugated to the anti-OA-3 mAb, OV-TL16. This conjugation strategy was designed to form a lysosomally degradable linkage that efficiently releases GA intracellularly. The linkage of 17APA-GA to HPMA significantly reduced the antiproliferative activity, with a 40-fold increase of the IC50 value (19 µM in A2780 cells) and the final product (anti-OA3:GA immunoconjugate) had modest activity, similar to the nonspecific conjugate of GA with bovine serum albumin. H:ABA-GA and H:APA-GA were then tested for their in vivo efficacy in a xenograft model of MDA-361/DYT2 tumors that were transplanted sc into athymic mice. Therapy was initiated when tumors had reached a detectable size (15-20 mm3) as described in the Experimental Procedures. The administration regimen was chosen to mimic a dose used in patients (4 mg/Kg) and corresponded to t1/2 (17) (i.e., ip injections every 3 days through 120 days). Efficacy was determined on the basis of survival and tumor progression. As shown in Figure 5, the survival of Herceptin-treated recipients was prolonged compared with that of the placebo group. The H:ABA-GA and H:APA-GA conjugates prolonged survival even further, but neither Herceptin nor the H:ABA-GA were able to induce a complete, stable tumor regression. However, treatment with the H:APA-GA immunoconjugate induced permanent tumor regression in 2/8 recipients. No tumors were detectable by visual inspection, even after 4 months had elapsed, 1 month after the ip injection treatment had been halted. Thus, H:APA-GA was qualitatively superior to H:ABA-GA because this immunoconjugate could cure a fraction of the recipient mice while H:ABA-GA, in repeated experiments, was only able to retard tumor growth. In this study, we have demonstrated that the strategy of BOC protecting and acid deprotection allowed production of large quantities of the desired 17-GMB-APA-GA in excellent purity. Thus, the previously encountered technical obstacles to larger-scale production of 17-APAGA immunoconjugates have been eliminated. Conjugation of 17-GMB-APA-GA to the anti-HER2 mAb Herceptin resulted in an immunoconjugate with enhanced

Modifications in Synthesis of Immunoconjugates

antiproliferative action when compared with that of the native mAb. More importantly, this immunoconjugate is superior to the previously studied immunoconjugate, H:ABA-GA, and shows significantly improved efficacy in vivo. ACKNOWLEDGMENT

The authors thank Robert Cohen and Mark Sliwkowski, Genentech Inc., for their generous supply of Herceptin, and the Laboratory of Analytical Chemistry, NIDDK, for their assistance in obtaining detailed mass spectra data for all of the geldanamycin derivatives. LITERATURE CITED (1) DeBoer, C., Meulman, P. A., Wnuk, R. J., and Peterson, D. H. (1970) Geldanamycin, a new antibiotic. J. Antibiot. 23, 442-447. (2) Neckers, L., Schulte, T. W., and Mimnaugh, E. (1999) Geldanamycin as a potential anti-cancer agent: Its molecular target and biochemical activity. Invest. New Drugs 17, 361373 (3) Sausville, E. A. (2001) Combining Cytotoxics and 17Allylamino, 17-Demethoxygeldanamycin: Sequence and Tumor Biology Matters. Clin. Cancer Res. 7, 2155-2158. (4) Schnur, R. C., Corman, M. L., Gallaschun, R. J., Cooper, B. A., Dee, M. F., Doty, J. L., Muzzi, M. L., Moyer, J. D., DiOrio, C. I., Barbacci, E. G., Miller, P. E., O’Brien, A. T., Motin, M. J., Foster, B. A., Polack, V. A., Savage, D. M., Sloan, D. E., Pustilnik, L. R., and Moyer, M. P. (1995) Inhibition of the Oncogene Product p185erbB-2 in Vitro and in Vivo by Geldanamycin and Dihydrogeldanamycin. J. Med. Chem. 38, 38063812. (5) Wilson, R. H., Takimoto, C. H., Agnew, E. B., Morrison, G., Grollman, F., Thomas, R. R., Saif, M. W., Hopkins, J., Allegra, C., Grochow, L., Szabo, E., Hamilton, J. M., Monahan, B. P., Neckers L., and Grem, J. L. (2001) Phase I pharmacologic study of 17-(allylamino)-17-demethoxygeldanamycin (AAG) in adult patients with advanced solid tumors. Proc. Am. Soc. Clin. Oncol., 37th Annual Meeting, San Francisco, CA, May 11-16, 2001. (6) Munster, P. N., Tong, W., Schwartz, L., Larson, S., Kenneson, K., De La Cruz, A., Rosen, N., and Scher, H. (2001) Phase I trial of 17-(allylamino)-17-demethoxygeldanamycin (17AAG) in patients with advanced solid malignancies. Proc. Am. Soc. Clin. Oncol., 37th Annual Meeting, San Francisco, CA, May 11-16, 2001. (7) Preijers, F. W. M. B., Tax, W. J. M., De Witte, T., Janssen, A., Heijden, H. V. D., Vidal, H., Wessels, J. M. C., and Capel, P. J. A. (1988) Relationship between internalization and cytotoxicity of ricin A-chain immunotoxins. Br. J. Haematol. 70, 289-294. (8) King, D. J. (1998) Applications and Engineering of Monoclonal Antibodies, Chapter 4, pp 119-160, Taylor and Francis Inc., Philadelphia, PA. (9) Tolcher, A. W., Ochoa, L., Patnaik, A., Hammond, L., Hildalgo, M., Edwards, T., Drengler, R., Erickson, J., DeWitte, M., Desai, K., Chari, R., Lambert, J., and Rowinsky, E. K. (2001) SB-408075, a tumor-activated prodrug maytansinoid immunoconjugate directed to the C242 antigen: a phase I pharmacokinetic and biologic correlative study. Proc. Am. Soc. Clin. Oncol., 37th Annual Meeting, San Francisco, CA, May 11-16, 2001. (10) Frankel, A. E., Sievers, E. L., and Scheinberg, D. A. (2000) Cell Surface Receptor-Targeted Therapy of Acute Myeloid Leukemia: A Review. Cancer Biother. Radiopharm. 15, 459476. (11) Baselga, J. (2001) Clinical Trials of Herceptin. Eur. J. Cancer 37, S18-S24. (12) Slamon, D. J., Clark, G. M., Wong, S. G., Levin, W. J., Ullrich, A., and McGuire, W. L. (1987) Human Breast

Bioconjugate Chem., Vol. 13, No. 4, 2002 791 Cancer: Correlation of Relapse and Survival with Amplification of the HER-2/neu Oncogene. Science 235, 177-182. (13) Muss, H. B., Thor, A. D. Berry, D. A., Kute, T., Liu, E. T., Koerner, F., Cirrincione, C. T., Budman, D. R., Wood, W. C., Barcos, M., and Henderson, I. C. (1994) c-erbB-2 Expression and Response to Adjuvant Therapy in Women with NodePositive Early Breast Cancer. N. Engl. J. Med. 330, 12601266. (14) Zheng, F. F., Kuduk, S. D., Chiosis, G., Munster, P. N., Sepp-Lorenzino, L., Danishefsky, S. J., and Rosen, N. (2000) Identification of a geldanamycin dimer that induces the selective degradation of HER-family tyrosine kinases. Cancer Res. 60, 2090-2094. (15) Mandler, R, Dadachova, E., Brechbiel, J. K., and Brechbiel, M. W. (2000) Synthesis and Evaluation of Anti-Proliferative Activity of a Geldanamycin-Herceptin Immunoconjugate. Bioorg. Med. Chem. Lett. 10, 1025-1028. (16) Mandler, R., Wu, C., Sausville, E. A., Roettinger, A. J., Newman, D. J., Ho, D. K., King, C. R., Yang, D., Lippman, M. E., Landolphi, N. F., Dadachova, E., Brechbiel, M. W., and Waldmann, T. A. (2000) Immunoconjugates of Geldanamycin and Anti-HER2 Monoclonal Antibodies: Antiproliferative Activity on Human Breast Carcinoma Cell Lines. J. Natl. Cancer Inst. 92, 1573-1581. (17) Mandler, R., Hinson, E. R., Davis, M. Y., Sausville, E. A., Newman, D. J., Yang, D., Roettinger, A. J., Brechbiel, M. W., and Waldmann, T. A. (2001) Herceptin: geldanamycin immunoconjugates augment the efficacy of Herceptin in mice bearing HER2-overexpressing xenografts. Clin. Cancer Res. 7, 3786S-3786S. (18) Kitagawa, T., Kawasaki, T., and Munechik, H. (1982) Enzyme immunoassay of blasticidin S with high selectivity: A new and convenient method for preparation of immunogenic (hapten-protein) conjugates. J. Biochem. 92, 585-590. (19) Yang, D., Kuan, C. T., Payne, J., Kihara, A., Murray, A., Wang, L. M., Alimandi, M., Pierce, J. H., Pastan, I., and Lippman, M. E. (1998) Recombinant heregulin-Pseudomonas exotoxin fusion proteins: interactions with the heregulin receptors and antitumor activity in vivo. Clin. Cancer Res. 4, 993-1004. (20) Heidaran, M. A., Fleming, T. P., Bottaro, D. P., Bell, G. I., Di Fiore, P. P., and Aaronson, S. A. (1990) Transformation of NIH3T3 fibroblasts by an expression vector for the human epidermal growth factor precursor. Oncogene 5, 1265-1270. (21) Kobayashi, H., Sakahara, H., Saga, T., Hosono, M., Shirato, M., Kanda, H., Ishibashi, K., Watanabe, T., Endo, K., Ishiwata, I., and Konishi, J. (1993) A human/mouse chimeric monoclonal antibody against CA125 for radioimmunoimaging of ovarian cancer. Cancer Immunol. Immunother. 37, 143149. (22) Kobayashi, H., Le, N., Kim, I. S., Kim, M.-K., Pie, J.-E., Drumm, D., Paik, D. S., Waldmann, T. A., Paik, C. H., and Carrasquillo, J. A. (1999) The pharmacokinetic characteristics of glycolated humanized anti-Tac Fabs are determined by their isoelectric points. Cancer Res. 59, 422-430. (23) Carter, P., Presta, L., Gorman, C. M., Ridgway, J. B. B., Henner, D., Wong, W. L. T., Rowland, A. M., Kotts, C., Carver, M. E., and Shepard, H. M. (1992) Humanization of an antip185/HER2 antibody for human cancer therapy. Proc. Natl. Acad. Sci. U.S.A. 89, 4285-4289. (24) Kasuya, Y., Lu, Z.-R., Kopeckova, P., and Kopecek, J. (2001) Improved synthesis and evaluation of 17-substituted aminoalkylgeldanamycin derivatives applicable to drug delivery systems Bioorg. Med. Chem. Lett. 11, 2089-2091. (25) Kasuya, Y., Lu, Z.-R., Kopeckova, P., Minko, T., Tabibi, S. E., and Kopecek, J. (2001) Synthesis and characterization of HPMA copolymer-aminopropylgeldanamycin conjugates. J. Controlled Release 74, 203-211.

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