Monoclonal Antibody Conjugates of Doxorubicin Prepared with

Feb 24, 1999 - Immunoconjugates of monoclonal antibody BR96 and Doxorubicin have been prepared using a novel series of branched hydrazone linkers. ...
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Bioconjugate Chem. 1999, 10, 279−288

279

Monoclonal Antibody Conjugates of Doxorubicin Prepared with Branched Linkers: A Novel Method for Increasing the Potency of Doxorubicin Immunoconjugates H. Dalton King,*,† Derek Yurgaitis,† David Willner,† Raymond A. Firestone,†,‡ Michael B. Yang,§ Shirley J. Lasch,§ Karl Erik Hellstro¨m,| and Pamela A. Trail§ Bristol Myers Squibb Pharmaceutical Research Institute, 5 Research Parkway, Wallingford, Connecticut 06492, and Princeton, New Jersey, 08543. Received August 28, 1998; Revised Manuscript Received December 28, 1998

Immunoconjugates of monoclonal antibody BR96 and Doxorubicin have been prepared using a novel series of branched hydrazone linkers. Since each linker bound to the mAb carries two DOX molecules, the DOX/mAb molar ratios of these conjugates were approximately 16, twice that of those previously prepared with single-chain hydrazone linkers. The conjugates were stable at a physiological pH of 7, but released DOX rapidly at lysosomal pH 5. The branched series of BR96 conjugates demonstrated antigen-specific cytotoxicity, and were more potent in vitro than the single-chain conjugate on both a DOX (4-14-fold) and mAb (7-23-fold) basis. The results suggest that, by using the branched linker methodology, it is possible to significantly reduce the amount of mAb required to achieve antigenspecific cytotoxic activity. In this paper, the synthesis and in vitro biology of branched chain immunoconjugates are described.

Monoclonal antibody conjugates have been shown to be effective targeting vehicles for cytotoxic drugs such as doxorubicin (1). We have previously demonstrated the antitumor activity of BR96-DOX conjugates in the treatment of human tumor xenografts in mice and rats (2, 3). The BR96 mAb identifies a Lewis y (Ley)-related tumorassociated antigen expressed at high density on the majority of human carcinomas (4). BR96 is rapidly internalized following antigen-specific binding (4, 5). The BR96-DOX conjugates employed a hydrazone linkage to C-13 of DOX (6-8) and a thioether linkage to the mAb (2, 9). Hydrazones of this nature are stable at normal physiological pH 7, but once internalized into the acidic compartment of endosomes/lysosomes, hydrolysis occurs liberating free DOX (6, 7). The thioether linkage is formed by the reaction of BR96 thiol groups, generated by mild DTT reduction of four interchain disulfides, with the linker 1. Therefore, the observed molar ratio (DOX/ mAb) of these first generation BR96 conjugates was typically 8 (2, 9). BR96-DOX conjugates produced antigenspecific antitumor activity and were more potent, were more active, and produced less systemic toxicity than unconjugated DOX in vivo (2, 3, 9). We have previously demonstrated that it is possible to increase the potency of lysine-linked SPDP-hydrazone conjugates by increasing the DOX/mAb molar ratio from 1 to 8 (10). When the molar ratio was increased beyond 8, significant losses in antigen-specific binding were * To whom correspondence should be addressed. E-mail: [email protected]. † Bristol Myers Squibb Pharmaceutical Research Institute, Wallingford, CT. ‡ Current address: Boehringer Ingelheim Pharmaceuticals, Ridgefield, CT. § Bristol Myers Squibb Pharmaceutical Research Institute, Princeton, NJ. |Original address: Bristol Myers Squibb Pharmaceutical Research Institute, Seattle, WA. Current address: Pacific Northwest Research Foundation, Seattle, WA.

observed. A similar loss in both binding avidity and antigen-specific potency was observed by other investigators as molar ratios of directly linked DOX conjugates exceeded 10 (11). In the studies described here we evaluated the potential to increase the potency of BR96-DOX conjugates by attaching more than one drug per thiol group generated on the mAb. This approach employs a branched linker (Scheme 1), carrying two hydrazide functional groups for the attachment of DOX. With the single branch point, MR’s are expected to approach 16, twice that of the prototype conjugate BR96-DOX. For these conjugates to be effective, they must maintain binding avidity and be capable of undergoing acid-catalyzed hydrolysis at rates similar to that of BR96-DOX (9). For the branched linkers 2 and 3, we used L-glutamic acid as the synthetic template to attach the required functionality. The two carboxyl groups were converted to hydrazides for DOX attachment, and the amino group was extended to a maleimide for mAb thiol group attachment. The length of the maleimide tether was varied (n ) 2, 3, and 5) to evaluate solubility and other subtle aspects of conjugate synthesis. To avoid potential crowding of the DOX moieties, we also synthesized an extended series 3 in which a β-alanyl unit was inserted at each glutamic acid carboxyl group. Finally, to evaluate the susceptibility of a typical branched linker to enzymatic degradation, we synthesized a D-glutamic acid analogue of 2b (Schemes 2 and 3). EXPERIMENTAL PROCEDURES

General. All reactions utilizing nonprotic solvents were carried out under inert N2 atmosphere. Reaction solvents were freshly opened Aldrich “Sure-Seal” quality. All other solvents and reagents were reagent grade and used without further purification. Bio-Beads SM-2 chromatographic support was supplied by Bio-Rad Laboratories. 1H NMR and 13C NMR spectra were obtained at

10.1021/bc980100i CCC: $18.00 © 1999 American Chemical Society Published on Web 02/24/1999

280 Bioconjugate Chem., Vol. 10, No. 2, 1999 Scheme 1. Branched DOX Hydrazone Linkers

King et al. Scheme 2. Synthesis of Series 2

SYNTHESIS

300 and 75 MHz, respectively, in the indicated solvents. Internal referencing was used and chemical shifts are reported in ppm. FTIR’s were obtained as KBr pellets. Melting points are uncorrected. Elemental analyses were provided by Oneida Research Services. TLC was carried out on Analtech silica gel GHLF plates, generally with 10-20% MeOH in methylene chloride as the eluting solvent, and visualized with UV light and chemical staining. Flash chromatography was carried out on Merck silica gel 60 (230-400 mesh). Routine HPLC of synthetic compounds was performed on a Perkin-Elmer 410 Pump and LC-235 Detector. HPLC columns were supplied by Phenomenex (IB-Sil 5 C-18). All compounds were homogeneous by TLC and/or HPLC. HPLC Analysis of Conjugates. SEC-HPLC analysis of conjugates was carried out on a Toyo Soda 5u TSK column (3000SW XL, 300 mm × 7.5 mm) equipped with a 5u TSK (3000SW XL, 40 mm × 6.0 mm) guard column. Samples were eluted at a flow rate of 1.0 mL/min with isocratic 0.2 M KH2PO4, 0.9% NaCl, pH 6.8, and monitored at 280 and 495 nm. HIC-HPLC analysis was carried out on a Biorad Biogel TSK-phenyl-5-PW column (75 mm × 7.5 mm). Samples were eluted under a 35 min gradient of 100% buffer A to 100% buffer B at 1.0 mL/min. Buffer A was 2 M NaCl, 25 mm NaH2PO4 (pH 7.0). Buffer B was 80% 50 mM NaH2PO4 (pH 7.0), 10% 2-propanol, and 10% acetonitrile. Eluant was monitored at 280 and 495 nm.

Z-Glutamyldi(Boc)hydrazide 4. Z-Glutamic acid (42.20 g, 150 mmol) and N-hydroxysuccinimide (34.53 g, 300 mmol) were dissolved in 150 mL of DMF at 0 °C under dry N2. A 0.5 M solution of dicyclohexylcarbodiimide in methylene chloride (600 mL, 300 mmol) was added dropwise over a 1 h period with stirring. The reaction was stored at 4 °C in the refrigerator for 18 h. Dicyclohexylurea precipitate (65.48 g, 98%) was filtered, and the filtrate was added directly to solid tert-butylcarbazate (39.65 g, 300 mmol). After being stirred at room temperature for 48 h, the reaction was rotary evaporated to an oil, which was redissolved in 300 mL of ethyl acetate/200 mL of ether. The organic layer was extracted three times with 200 mL of 10% citric acid, 3 times with 200 mL of saturated aqueous sodium bicarbonate, and once with 100 mL of brine. The organic layer was dried over sodium sulfate and rotary evaporated to a foam. Flash chromatography was carried out on silica gel (4 in. × 19 in.) with ethyl acetate-hexane 2:1, 12 L. Pure fractions containing product 4 were pooled and concentrated to a foam by rotary evaporation to yield, after drying under high vacuum, 55.24 g (72%). 1 H NMR (CDCl3): δ 1.44 and 1.47 (2s, 18H), 1.9-2.4 (bm, 4H), 4.32 (bm, 1H), 5.06 (dd, J ) 12, 16 Hz, 2H), 5.55 (d, J ) 6 Hz, 1H), 6.5 (bd, 2H), 7.31 (bm, 5H), 9.6 (s, 1H), and 9.9 (s, 1H). Mass Spec.: FAB 510 (M + H+), 532 (M + Na+), 548.1 (M + K+). Anal.: C23H35N5O8 C, H, N. Z-(D)-Glutamyldi(Boc)hydrazide D-4. This compound was prepared on an identical scale by a method

Monoclonal Antibody Conjugates of Doxorubicin

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Scheme 3. Synthesis of Series 3

similar to that used for 4. Flash chromatography was carried out on silica gel (4 in. × 18 in.) with the following gradient: (1) CH2Cl2, 2 L, (2) CH2Cl2-methanol 25:1, 4 L, and (3) CH2Cl2-methanol 9:1, 6 L. Yield of D-4 was 59.11 g (77%). 1H NMR (CDCl ): δ 1.44 and 1.47 (2s, 18H), 1.9-2.4 3 (bm, 4H), 4.32 (bm, 1H), 5.06 (dd, J ) 12, 16 Hz, 2H), 5.57 (d, J ) 7 Hz, 1H), 6.6 (m, 2H), 7.31 (bm, 5H), 9.60 (s, 1H), and 9.87 (s, 1H). Mass Spec.: FAB 532 (M + Na+), 549 (M + K+). Anal.: C23H35N5O8 C, H, N. Glutamyldi(Boc)hydrazide 5. Z-Glutamyldi(Boc)hydrazide 4 (19.59 g, 38.44 mmol) was hydrogenated along with 2 g of 10% Pd-C in 200 mL of MeOH at 50 psi for 3 h. The reaction was filtered through Celite and rotary evaporated. The resulting foam was dried under high vacuum to yield 5 (14.40 g, 100%). 1H NMR (d -methanol): δ 1.42 and 1.45 (2s, 18H), 1.9 4 (bm, 2H), 2.35 (t, J ) 7 Hz, 2H), 3.34 (t, 7 Hz, 1H). Mass Spec.: DCI 376 (M + H)+. Anal.: C15H29N5O6‚0.5H2O C, H, N. (D)-Glutamyldi(Boc)hydrazide D-5. This compound was prepared from D-4 (23.05 g, 45.2 mmol) by an similar procedure to that used for 5. Crude product was purified by flash chromatography on silica gel (2 in. × 20 in.) with the following gradient: (1) CH2Cl2-methanol 25:1, 600 mL, (2) CH2Cl2-methanol 9:1, 6 L, and (3) CH2Cl2methanol 8:2, 4 L. Pure fractions were pooled and rotary evaporated. Drying under high vacuum yielded pure D-5 (13.51 g, 80%). 1H NMR (d -methanol): δ 1.46 and 1.47 (2s, 18H), 1.94 4 (bm, 2H), 2.33 (t, J ) 7 Hz, 2H), 3.34 (t, J ) 7 Hz, 1H). Mass Spec.: FAB 376 (M + H)+, 398 (M + Na)+, 414 (M + K)+. Anal.: C15H29N5O6‚0.5H2O C, H, N. Maleimidopropionylglutamyldi(Boc)hydrazide 6a. Maleimidopropionic acid (636 mg, 3.76 mmol) and Nhydroxysuccinimide (476 mg, 4.14 mmol) were dissolved in 10 mL of DMF at 0 °C. A 0.5 M solution of DCC in CH2Cl2 (7.6 mL, 3.8 mmol) was added and the reaction allowed to stand for 20 h at 4 °C. After filtration of the

DCU precipitate, the filtrate was added to 5 (1.27 g, 3.38 mmol) and the mixture was stirred at room temperature for 2.5 days. Solvents were partially removed by rotary evaporation. The oil was dissolved in 100 mL of ethyl acetate, then extracted three times with 100 mL of 10% citric acid, three times with 100 mL of saturated aqueous sodium bicarbonate, and three times with 100 mL of H2O. The organic layer was dried over sodium sulfate and rotary evaporated to a foam. This was purified by flash chromatography on silica gel (2 in. × 11 in.) with CH2Cl2-acetic acid-methanol 93:2:5. Pure fractions were pooled, rotary evaporated, and dried under high vacuum to yield 6a as a foam (1.22 g, 69%). 1 H NMR (d4-methanol): δ 1.46 (s, 18H), 2.01 (m, 2H), 2.33 (t, 7 Hz, 2H), 2.51 (t, J ) 7 Hz, 2H), 3.76 (t, J ) 7 Hz, 2H), 4.34 (t, J ) 7 Hz, 1H), 6.80 (s, 2H). Mass Spec.: FAB 549.4 (M + Na)+, 565.3 (M + K)+. Anal.: C22H34N6O9‚ 2HOAc C, H, N. Maleimidobutyrylglutamyldi(Boc)hydrazide 6b. This compound was prepared by a method analagous to that used for 6a from maleimidobutyric acid (1.9 g, 10.3 mmol). Crude product was purified through a plug of silica gel with CH2Cl2-acetic acid-methanol 93:2:5, rotary evaporated, and dried under high vacuum to yield 6b as a foam (3.50 g, 63%). 1 H NMR (d4-methanol): δ 1.36 and 1.37 (2s, 18H), 1.77 (p, J ) 7 Hz, 2H), 2.00 (bm, 2H), 2.14 (t, J ) 7 Hz, 2H), 2.26 (t, J ) 7 Hz, 2H), 3.43 (t, J ) 7 Hz, 2H), 4.26 (t, J ) 7 Hz, 1H), 6.71 (s, 2H). Mass Spec.: 541 (M + H)+, 563 (M + Na)+, 579 (M + K)+. Anal.: C23H36N6O9‚ 0.75H2O C, H, N. Maleimidobutyryl-(D)-glutamyldi(Boc)hydrazide D-6b. Maleimidobutyric acid (1.832 g, 10.0 mmol) was dissolved with N-methylmorpholine (1.21 mL, 11.0 mmol) in 60 mL of dry THF under N2 at 0 °C. Isobutylchloroformate (1.30 mL, 10.0 mmol) was added dropwise, followed 10 min later by the addition of (D)-Glutamyldi(Boc)hydrazide (D-5) (3.754 g, 10.0 mmol). Stirring was continued for 1 h at 0 °C. The reaction was rotary

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evaporated to a foam, which was then dissolved in 150 mL of EtOAc. The organic layer was washed two times with 100 mL of 10% citric acid and two times with 100 mL of saturated NaHCO3. The organic layer was concentrated to a foam, which was purified by flash chromatography on silica gel (2 in. × 11 in.) with CH2Cl2acetic acid-methanol 95:2:3, 2 L, followed by CH2Cl2acetic acid-methanol 93:2:5, 1 L. Pure fractions were pooled and rotary evaporated to a foam. Drying under high vacuum yielded 6b (3.25 g, 60%). 1H NMR (d -methanol): δ 1.45 and 1.46 (2s, 18H), 1.86 4 (p, J ) 7 Hz, 2H), 2.09 (bm, 2H), 2.24 (t, J ) 7 Hz, 2H), 2.35 (t, J ) 7 Hz, 2H), 3.52 (t, J ) 7 Hz, 2H), 4.35 (t, J ) 7 Hz, 1H), 6.81 (s, 2H). Mass Spec.: 563 (M + Na)+, 579 (M + K)+. Anal.: C23H36N6O9‚0.75H2O C, H, N. Maleimidocaproylglutamyldi(Boc)hydrazide 6c. This compound was prepared by a method analagous to that used for 6a from maleimidocaproic acid (4.22 g, 20 mmol). Crude product was purified by flash chromatography on silica gel (2 in. × 10 in.) with 4 L of CH2Cl2acetic acid-methanol 97:1:2. Pure fractions were pooled, rotary evaporated, and dried under high vacuum to yield 6c as a foam (6.40 g, 56%). 1 H NMR (d4-methanol): δ 1.2 (p, J ) 7 Hz, 2H), 1.40 (s, 18H), 1.5 (m, 4H), 2.0 (bm, 2H), 2.14 (t, J ) 7 Hz, 2H), 2.28 (t, J ) 7 Hz, 2H), 3.41 (t, J ) 7 Hz, 2H), 4.29 (t, J ) 7 Hz, 1H), 6.72 (s, 2H). Mass Spec.: FAB 569 (M + H)+, 591 (M + Na)+, 607 (M + K)+. Anal.: C25H40N6O9‚ 0.5H2O C, H, N. Maleimidopropionylglutamyldihydrazide Ditrifluoroacetate 7a. Maleimidopropionylglutamyldi(Boc)hydrazide 6a (1.50 g, 2.85 mmol) was stirred in 15 mL of CH2Cl2/trifluoroacetic acid (1:1) under N2 for 1.5 h. Solvents were removed by rotary evaporation. Ether was added and coevaporated three times, and then the resulting solid was triturated with ether. The solid was filtered and dried under high vacuum to yield 7a (1.6 g, 100%). 1 H NMR (d4-methanol): δ 1.99 and 2.16 (2m, 2H), 2.41 (t, J ) 7 Hz, 2H), 2.53 (t, J ) 7 Hz, 2H), 3.80 (dt, J ) 2, 7 Hz, 2H), 4.38 (dd, J ) 5, 4 Hz, 1H), 6.81 (s, 2H). Mass Spec.: FAB 349.2 (M + Na)+, 365.1 (M + K)+. Anal.: C12H18N6O5‚2.8TFA C, H, N. Maleimidobutyrylglutamyldihydrazide Ditrifluoroacetate 7b. This compound was prepared by a similar procedure from 6b (3.50 g, 6.47 mmol). Yield of 7b was 3.8 g (100%). 1 H NMR (d4-methanol): δ 1.87 (p, J ) 7 Hz, 2H), 2.0 and 2.2 (2m, 2H), 2.27 (t, J ) 7 Hz, 2H), 2.44 (m, 2H), 3.53 (t, J ) 7 Hz, 2H), 4.42 (dd, J ) 5, 4 Hz, 1H), 6.82 (s, 2H). Mass Spec.: FAB 341 (M + H)+, 363 (M + Na)+, 379 (M + K)+. Anal.: C13H20N6O5‚3.15TFA C, H, N. Maleimidobutyryl-(D)-glutamyldihydrazide Ditrifluoroacetate D-7b. This compound was prepared by a similar procedure from D-6b (2.06 g, 3.81 mmol). Yield of D-7b was 2.2 g (100%). 1H NMR (d -methanol): δ 1.79 (p, J ) 7 Hz, 2H), 1.9 4 and 2.1 (2m, 2H), 2.18 (t, J ) 7 Hz, 2H), 2.37 (m, 2H), 3.45 (t, J ) 7 Hz, 2H), 4.35 (dd, J ) 5, 4 Hz, 1H), 6.73 (s, 2H). Mass Spec.: FAB 341 (M + H)+, 363 (M + Na)+, 379 (M + K)+. Anal.: C13H20N6O5‚2.5TFA C, H, N. Maleimidocaproylglutamyldihydrazide Ditrifluoroacetate 7c. This compound was prepared by a similar procedure from 6c (5.96 g, 10.5 mmol). Yield of 7c was 6.3 g (100%). 1H NMR (d -methanol): δ 1.22 (p, J ) 7 Hz, 2H), 1.52 4 (m, 4H), 1.92 and 2.09 (2m, 2H), 2.18 (t, J ) 7 Hz, 2H), 2.35 (m, 2H), 3.41 (t, J ) 7 Hz, 2H), 4.35 (dd, J ) 5,4 Hz,

King et al.

1H), 6.72 (s, 2H). Mass Spec.: FAB 369 (M + H)+, 391 (M + Na)+, 407 (M + K)+. Anal.: C15H24N6O5‚2.5TFA C, H, N. MaleimidopropionylglutamyldihydrazoneofDoxorubicin 2a. Maleimidopropionylglutamyldihydrazide ditrifluoroacetate 7a (600 mg, 1.07 mmol) and DOX‚HCl (1.24 g, 2.14 mmol) were dissolved in 600 mL of methanol over a period of 3 h. The reaction was concentrated to 100 mL by rotary evaporation, then stirred for 3 days. The reaction was further concentrated to 12 mL and eluted on an LH-20 column (2 in. × 10 in.) with methanol. Chromatography was repeated in the same system on mixed fractions. The purified product was rotary evaporated to a red film and dried under high vacuum to yield 2a (776 mg, 50%). 1H NMR (d -methanol): (selected peaks) δ 1.34 (2d, J 4 ) 7 Hz, 6H), 4.07 (2s, 6H), 6.79 (s, 2H), 7.5-8.0 (m, 6H). Mass Spec.: FAB 1375.4 (M - H)-. Ionspray: 1377.2 (MH+). HRMS: calculated (M + Na)+ 1399.4506; observed 1399.4455. Anal.: C66H72N8O25‚2HCl‚3.0H2O C, H, N. Maleimidobutyrylglutamyldihydrazone of Doxorubicin 2b. This compound was prepared by a similar procedure from 7b (1.00 g, 1.76 mmol) and DOX‚HCl (2.05 g, 3.53 mmol). Yield of 2b was 1.32 g (51%). 1H NMR (d -methanol): (selected peaks) δ 1.33 (2d, J 4 ) 7 Hz, 6H), 4.06 (2s, 6H), 6.80 (s, 2H), 7.5-8.0 (m, 6H). Mass Spec.: FAB 1392 (MH+), 1413.4 (M + Na)+, 1429 (M + K)+. Ionspray: 1392.5 (M + H)+, 1414.4 (M + Na)+. HRMS: calculated (M + Na)+ 1413.4663; observed 1413.4609. Anal.: C67H74N8O25‚2HCl‚4.0H2O C, H, N. Maleimidobutyryl-(D)-glutamyldihydrazone of Doxorubicin D-2b. This compound was prepared by a similar procedure from D-7b (570 mg, 1.00 mmol) and DOX‚HCl (1.34 g, 2.30 mmol). Yield of D-2b was 420 mg (30%). 1 H NMR (d4-methanol): (selected peaks) δ 1.30 (2s, 6H), 4.07 (2s, 6H), 6.80 (s, 2H), 7.5-8.0 (m, 6H). Mass Spec.: FAB 1392.0 (MH+), 1414.9 (M + Na)+, 1429.7 (M + K)+. HRMS: calculated (M + Na)+ 1413.4663; observed 1413.4603. Anal.: C67H74N8O25‚2HCl‚3.5H2O C, H, N. Maleimidocaproylglutamyldihydrazone of Doxorubicin 2c. This compound was prepared by a similar procedure from 7c (298 mg, 0.50 mmol) and DOX‚HCl (580 mg, 1.00 mmol). Yield of 2c was 510 mg (68%). 1H NMR (d -methanol): (selected peaks) δ 1.34 (2s, 4 6H), 4.08 (2s, 6H), 6.76 (s, 2H), 7.5-8.0 (m, 6H). Mass Spec.: FAB 1420 (MH+), 1442.3 (M + Na)+. Ionspray: 1419.6 (M + H)+. HRMS: calculated (M + Na)+ 1441.4976; observed 1441.4924. Anal.: C69H78N8O25‚2HCl‚4H2O C, H, N. Z-β-Alanyl(BOC)hydrazide 8. Z-β-Alanine (8.93 g, 40 mmol), tert-butylcarbazate (5.29 g, 40 mmol), and EDCI (8.00 g, 42 mmol) were stirred in 200 mL of CH2Cl2 for 1.5 h at room temperature. The reaction was extracted three times with 200 mL of 0.1 M acetic acid, twice with 200 mL of saturated aqueous sodium bicarbonate, and once with 200 mL of water. The organic layer was dried over sodium sulfate, rotary evaporated, and dried under high vacuum to yield 8 as a foam, 12.42 g (92%). 1H NMR (d -DMSO): δ 1.38 (s, 9H), 2.25 (t, J ) 7 Hz, 6 2H), 3.19 (q, J ) 7 Hz, 2H), 4.99 (s, 2H), 7.3 (m, 6H), 8.21 (s, 1H), 9.56 (s, 1H). Mass Spec.: FAB 338 (M + H)+. Anal.: C16H23N3O5 C, H, N. β-Alanyl(BOC)hydrazide 9. 8 (15.25 g, 45.2 mmol) was hydrogenated at 50 psi in 200 mL of methanol with 3 g of 10% Pd-C for 4 h. The reaction was filtered

Monoclonal Antibody Conjugates of Doxorubicin

through Celite, rotary evaporated, and dried under high vacuum to yield 9 as a hygroscopic foam, 9.2 g (100%). 1H NMR (d -methanol): δ 1.40 (s, 9H), 2.32 (t, J ) 7 4 Hz, 2H), 2.88 (t, J ) 7 Hz, 2H). Mass Spec.: FAB 204.2 (M + H)+. Anal.: C8H17N3O3‚0.5H2O C, H, N. Z-Glutamyldi[β-alanyl(Boc)hydrazide] 10. Z-Glutamic acid (3.86 g, 13.7 mmol) and N-hydroxy succinimide (3.17 g, 27.5 mmol) were dissolved in 80 mL of DMF at 0 °C under dry N2. A 0.5 M solution of dicyclohexylcarbodiimide in methylene chloride (55 mL, 27.5 mmol) was added, and the reaction was stored at 4 °C for 24 h. Dicyclohexylurea precipitate was filtered, and the filtrate was added to 9 (6.00 g, 29.5 mmol). After the mixture was stirred at room temperature for 15 h, the reaction was rotary evaporated to an oil, which was redissolved in 150 mL of ethyl acetate. The organic layer was extracted three times with 100 mL of 10% citric acid, 3 times with 100 mL of saturated aqueous sodium bicarbonate, and three times with 100 mL of brine. The organic layer was dried over sodium sulfate and rotary evaporated to a foam. Flash chromatography was carried out on silica gel (2 in. × 11 in.) with 1 L of CH2Cl2/ methanol 25:1 followed by 3 L of CH2Cl2/methanol 9:1. Pure fractions containing product 10 were pooled and concentrated to a foam by rotary evaporation to yield, after drying under high vacuum, 6.70 g (75%). 1 H NMR (CDCl3): δ 1.42 (s, 18H), 2.03 and 2.32 (2m, 8H), 3.5 (m, 4H), 4.35 (t, J ) 7 Hz, 1H), 5.05 (dd, J ) 18 Hz, 2H), 6.22 (d, J ) 7 Hz, 1H), 6.49 (d, J ) 18 Hz, 2H), 7.30 (s, 5H), 7.42 (m, 1H), 7.58 (m, 1H). Mass Spec.: DCI 652 (M + H)+, 674 (M + Na)+, 690 (M + K)+. Anal.: C29H45N7O10 C, H, N. Glutamyldi[β-alanyl(Boc)hydrazide] 11. Z-Glutamyldi[β-alanyl(Boc)hydrazide] 10 (3.52 g, 5.40 mmol) was hydrogenated along with 1 g of 10% Pd-C in 75 mL of MeOH at 50 psi for 2 h. The reaction was filtered through Celite and rotary evaporated. The resulting foam was dried under high vacuum to yield 11 (2.77 g, 99%). 1H NMR (d -methanol): δ 1.46 (s, 18H), 1.91 (m, 2H), 4 2.25 (t, J ) 7 Hz, 2H), 2.42 (q, J ) 8 Hz, 4H), 3.35 (t, J ) 8 Hz, 1H), 3.44 (m, 4H). Mass Spec.: FAB 518 (M + H)+, 540 (M + Na)+, 556 (M + K)+. Anal.: C21H39N7O8‚ 1.5H2O C, H, N. Maleimidopropionylglutamyldi[β-alanyl(Boc)hydrazide] 12a. Maleimidopropionic acid (399 mg, 2.36 mmol) and N-hydroxy succinimide (272 mg, 2.36 mmol) were dissolved in 30 mL of CH2Cl2/3 mL of DMF at 0 °C. A 0.5 M solution of DCC in CH2Cl2 (4.7 mL, 2.36 mmol) was added and the reaction mixture stirred for 3 h at room temperature. After filtration of the DCU precipitate, the filtrate was added to 11 (1.10 g, 2.13 mmol) and the reaction mixture stirred at room temperature for 1 day. Solvents were removed by rotary evaporation. The oil was purified by flash chromatography on silica gel (2 in. × 10 in.) with 500 mL of CH2Cl2, 2 L of CH2Cl2/methanol 95:5, and 2 L of CH2Cl2/methanol 9:1. Pure fractions were pooled, rotary evaporated, and dried under high vacuum to yield 12a as a foam (850 mg, 60%). 1H NMR (d -methanol): δ 1.46 (s, 18H), 1.82 and 2.04 4 (2m, 2H), 2.23 (t, J ) 8 Hz, 2H), 2.40 (m, 4H), 2.52 (t, J ) 7 Hz, 2H), 3.45 (m, 4H), 3.78 (t, J ) 7 Hz, 2H), 4.20 (dd, J ) 8 Hz, 1H), 6.81 (s, 2H). Mass Spec.: FAB 669 (M + H)+, 691 (M + Na)+, 707 (M + K)+. Anal.: C28H44N8O11‚2H2O C, H, N. Maleimidobutyrylglutamyldi[β-alanyl(Boc)hydrazide] 12b. This compound was prepared by a similar procedure from maleimidobutyric acid (432 mg, 2.36 mmol). Yield of 12b was 800 mg (55%).

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NMR (d4-methanol): δ 1.46 (s, 18H), 1.87 (m, 3H), 2.08 (m, 1H), 2.24 (m, 4H), 2.41 (m, 4H), 3.45 (m, 6H), 4.23 (dd, J ) 8 Hz, 1H), 6.82 (s, 2H). Mass Spec.: FAB 683 (M + H)+, 705 (M + Na)+, 721 (M + K)+. Anal.: C29H46N8O11‚1.5H2O C, H, N. Maleimidocaproylglutamyldi[β-alanyl(Boc)hydrazide] 12c. Maleimidocaproic acid (453 mg, 2.14 mmol) and N-methylmorpholine (239 mg, 2.36 mmol) were dissolved in 25 mL of dry THF under Ar at -5 °C. Isobutylchloroformate (263 mg, 1.93 mmol) was added. After 5 min, 11 (1.0 g, 1.93 mmol) was added as a THF solution and the reaction mixture stirred for 3 h with warming to room temperature. Ethyl acetate (150 mL) was added, and then the solution was extracted three times with 75 mL of 10% citric acid, three times with 75 mL of saturated aqueous sodium bicarbonate, and three times with 75 mL of water. The organic layer was dried over sodium sulfate, then passed through a plug of silica gel with CH2Cl2/methanol 9:1. The purified product was rotary evaporated and dried under high vacuum to give 12c, 800 mg (58%). 1H NMR (d -methanol): δ 1.30 (m, 2H), 1.46 (s, 18H), 4 1.60 (m, 4H), 1.88 and 2.06 (2m, 2H), 2.22 (t, J ) 7 Hz, 4H), 2.41 (t, J ) 7 Hz, 4H), 3.44 (m, 6H), 4.24 (dd, J ) 9 Hz, 1H), 6.80 (s, 2H). Mass Spec.: FAB 711.4 (M + H)+, 733.2 (M + Na)+, 749.3 (M + K)+. Anal.: C31H50N8O11‚ 1.0H2O C, H, N. Maleimidopropionylglutamyldi[β-alanylhydrazide] 13a. Maleimidopropionylglutamyldi[β-alanyl(Boc)hydrazide] 12a (850 mg, 1.27 mmol) was stirred in 15 mL of CH2Cl2/trifluoroacetic acid (1:1) under N2 for 1.5 h. Solvents were removed by rotary evaporation. Ether was added and coevaporated three times, and then the resulting solid was triturated with ether. The solid was filtered and dried under high vacuum to yield 13a (890 mg, 100%). 1H NMR (d -methanol): δ 1.83 and 2.02 (2m, 2H), 2.23 4 (t, J ) 7 Hz, 2H), 2.52 (q, J ) 7 Hz, 6H), 3.47 (m, 4H), 3.78 (dt, J ) 7, 3 Hz, 2H), 4.13 (dd, J ) 5, 4 Hz, 1H), 6.82 (s, 2H). Mass Spec.: FAB 469.0 (M + H)+, 491.1 (M + Na)+, 507.1 (M + K)+. Anal.: C18H28N8O7‚3.75TFA‚ 0.25Et2O C, H, N. Maleimidobutyrylglutamyldi[β-alanylhydrazide] 13b. This compound was prepared by a similar procedure from 12b (800 mg, 1.17 mmol). Yield of 13b was 840 mg (100%). 1 H NMR (d4-methanol): δ 1.88 (m, 3H), 2.06 (m, 1H), 2.26 (t, J ) 7 Hz, 4H), 2.51 (t, J ) 7 Hz, 4H), 3.50 (m, 6H), 4.18 (dd, J ) 5, 4 Hz, 1H), 6.82 (s, 2H). Mass Spec.: FAB 483.2 (M + H)+, 505.1 (M + Na)+, 521.1 (M + K)+. Anal.: C19H30N8O7‚3.5TFA‚0.25Et2O C, H, N. Maleimidocaproylglutamyldi[β-alanylhydrazide] 13c. This compound was prepared by a similar procedure from 12c (800 mg, 1.13 mmol). Yield of 13c was 870 mg (100%). 1H NMR (d -methanol): δ 1.31 (p, J ) 7 Hz, 2H), 1.61 4 (m, 4H), 1.85 and 2.03 (2m, 2H), 2.24 (t, J ) 7 Hz, 4H), 2.50 (t, J ) 7 Hz, 4H), 3.47 (m, 6H), 4.19 (dd, J ) 5, 4 Hz, 1H), 6.80 (s, 2H). Mass Spec.: Ionspray 511.1 (M + H)+, 533.0 (M + Na)+. Anal.: C21H34N8O7‚2.75TFA‚ 0.25Et2O C, H, N. Maleimidopropionylglutamyldi[β-alanylhydrazone] of Doxorubicin 3a. Maleimidopropionylglutamyldi[β-alanylhydrazide] ditrifluoroacetate 13a (1.0 g, 1.44 mmol) and DOX‚HCl (1.68 g, 2.88 mmol) were dissolved in 600 mL of methanol over a period of 3 h. The reaction was concentrated to 100 mL by rotary evaporation, then stirred for 1 day. After further concentration to 10 mL, elution on an LH-20 column (2 in. × 10 in.) with 1H

284 Bioconjugate Chem., Vol. 10, No. 2, 1999

methanol/DMF (1:1) was carried out. The purified product was concentrated by rotary evaporation and precipitated by the addition of acetonitrile. The red solid was isolated by centrifugation and dried under high vacuum to yield 3a (450 mg, 20%). 1H NMR (d -methanol): δ 1.29 (2bd, 6H), 4.04 (s, 6H), 4 6.80 (s, 2H), 7.5-8.0 (m, 6H). Mass Spec.: Ionspray 1519.6 (M + H)+, 1541.2 (M + Na)+. HRMS: calculated (M + Na)+ 1541.5249; observed 1541.5187. Anal.: C72H82N10O27‚2HCl‚7H2O C, H, N. Maleimidobutyrylglutamyldi[β-alanylhydrazone] of Doxorubicin 3b. This compound was prepared by a similar procedure from 13b (280 mg, 0.395 mmol). Chromatography was carried out on an LH-20 column (1 in. × 15 in.) with methanol/DMF (1:1). The purified product was concentrated by rotary evaporation and precipitated by the addition of acetonitrile. The red solid was isolated by centrifugation and dried under high vacuum to yield 3b (325 mg, 51%). 1 H NMR (d4-methanol): δ 1.30 (m, 6H), 4.04 (s, 6H), 6.78 (s, 2H), 7.4-8.0 (m, 6H). Mass Spec.: FAB 1533.7 (M + H)+, 1555.5 (M + Na)+, 1572.4 (M + K)+. Anal.: C73H84N10O27‚2HCl‚7H2O C, H; N; calcd, 8.08; found, 7.41. Maleimidocaproylglutamyldi[β-alanylhydrazone] of Doxorubicin 3c. By using the procedure of 3b, we prepared this compound from 13c (148 mg, 0.20 mmol). Yield of 3c was 162 mg (50%). 1 H NMR (d6-DMSO): δ 1.20 (m, 6H), 4.0 (s, 6H), 6.95 (s, 2H), 7.5-8.1 (m, 6H). Mass Spec.: FAB 1561 (M + H)+, 1583.4 (M + Na)+, 1599.9 (M + K)+. Anal.: C75H88N10O7‚2HCl‚7H2O C, H, N. CONJUGATE SYNTHESIS

Monoclonal Antibodies. BR96 is a chimeric (mouse/ human IgG1) mAb which identifies a Ley-related tumorassociated antigen expressed on carcinomas of the lung, colon, breast, and ovary. The mAb is rapidly internalized following antigen-specific binding (2, 4, 9). Doxorubicin immunoconjugates of various DOX/mAb molar ratios were prepared with both chimeric BR96 and control human IgG. Thiolation. Method A. On a scale e3 g, BR96 and IgG were reduced by a previously reported method (9). In a typical example, 1.54 g of BR96 (180 mL at 53.4 µM, 9.6 µmol) was deoxygenated by cycling several times between vacuum and Ar atmosphere. This was then treated with 34 mM DTT (2.0 mL, 68.0 umol in Ar-bubbled PBS, pH 7.0) and stirred at 37 °C under Ar for 3 h. Removal of low molecular weight compounds was accomplished by ultrafiltration against PBS, pH 7.0, in an Amicon stirred cell at 4 °C. A 400 mL Amicon cell was fitted with an Amicon YM30 filter (molecular weight cutoff 30000) and charged to 40 psi with Ar. Cell eluant was monitored for thiol content with Ellman’s reagent (12) until a baseline reading at 412 nm was obtained. Concentrations of protein and thiol groups were determined according to the previously reported method (9). In this example, 1.47 g of reduced BR96 (190 mL at 48.57 µΜ mAb, 412.7 µΜ thiol) was obtained, for a yield of 95% and a thiol titer of 8.5 mol of thiol groups/mol of BR96. Method B. On a scale >3 g, the same procedure was utilized for the DTT reaction, with the exception that the mAb solutions were deoxygenated by bubbling with Ar. Purification after DTT reduction was accomplished by ultrafiltration in a Filtron Minisette unit. The Minisette was fitted with two Filtron 30K cassettes and was connected to a Watson Marlow 604S pump with Bioprene tubing. The mAb solution was ultrafiltered at 0 °C under

King et al. Scheme 4. Conjugation of Branched Linkers to mAb’s BR96 and IgG

Table 1. Representative Immunoconjugates SH conjugatea titer

MR

BR96-2a 7.9 15.6 IgG-2a 10.0 15.9 BR96-2b 8.5 15.1 IgG-2b 8.6 14.5 BR96-D-2b 8.4 15.3 IgG-D-2b 8.6 15.4 BR96-2c 7.2 14.6 IgG-2c 9.9 16.2 BR96-3a 7.9 11.7 IgG-3a 9.4 14.7 BR96-3b 7.9 11.9 IgG-3b 9.9 16.2 BR96-3c 6.8 13.9

yield aggregate unconjugated unreacted (%) (%) DOX (%)b mAb (%) 94 87 100 100 41e 57 96 86 100 71 100 75 50e

4.5 3.1 0.5 300 >160 >235 >300 >300 450 >415 >300 >300 >85 >300 >117

15b

19

>400

15c

14

>420

16b

1.5

120

16c

2.5

60

a 100 mM NaOAc. b 100 mM Na HPO . c MAb-linker; Linkers 2 4 and conjugates defined in Schemes 1 and 4.

(2-3) and treated for 10 min with 100 mL of 2-mercaptoethanol. Solvent was removed under vacuum and the residue washed with Et2O. Complete conversion to the thioether was verified by 1H NMR and a shift in HPLC retention time. Determination of Hydrolysis Kinetics of 14-16. Stock solutions (10 mM) of 14-16 were freshly prepared in H2O. Aliquots were diluted to a final concentration of 30 mM in either 100 mM NaOAc, pH 5.0, or 100 mM Na2HPO4, pH 7.0, then incubated (stoppered test tube) at 37 °C in a thermostat-regulated H2O bath. RP-HPLC was used to monitor the formation of DOX in solution and the disappearance of starting materials until equilibrium was reached. Conditions for the analyses were 50 mL injection; Phenomenex IB-Sil 5 C-18 column; mobile phase 44:56 H2O-MeOH with 6% NH4OAc; flow rate 1.5 mL/min; UV detection 235 nm. Free DOX was quantitated by integrating the 7.5 min peak (positively identified by comparison to a DOX standard) and represented as the percent of total DOX in solution vs time. Half-lives for the appearance of DOX were calculated from these curves and are listed in Table 2. Determination of Conjugate Hydrolysis Kinetics. Conjugates at approximately 10 mg/mL were diluted 1:3 into either 100 mM NaOAc, pH 5.0, or 100 mM Na2HPO4, pH 7.0. The solutions were placed into a temperaturecontrolled (37 °C) autosampler for RP-HPLC. Samples were taken and analyzed automatically at timed intervals by the autosampler. Conditions for the analyses were Jones Chromatography Apex C-18 5µ column; mobile phase 70:30 MeOH-30mM ammonium phosphate, pH 4.5; flow rate 1.5 mL/min; fluorescence exitation 233 nm, emission 570 nm; UV detection 235 nm. DOX‚HCl standards were analyzed before and after the experiment. The results are expressed as percents of total DOX released. Half-lives for the appearance of DOX were calculated from these curves and are listed in Table 2. In Vitro Cytotoxicity Assays. In vitro cytotoxicity assays were performed as described previously (10) using the BR96 expressing human lung carcinoma line L2987 (2). Briefly, monolayer cultures of L2987 cells were harvested and resuspended to 1 × 105/mL in RPMI-1640 containing 10% heat inactivated fetal calf serum (RPMI10%FCS). Cells (0.1 mL/well) were added to each well of 96 well microtiter plates and incubated overnight at 37 °C in a humidified atmosphere containing 5% CO2. Medium was removed from the plates and serial dilutions of DOX or mAb-DOX conjugates added to the wells. All

dilutions were performed in quadruplicate. Cells were exposed to DOX or mAb-DOX conjugates for 2 h at 37 °C in a humidified atmosphere of 5% CO2. Plates were centrifuged (200g, 5 min), the drug or conjugate removed, and the cells washed 3× with RPMI-10% FCS. The cells were cultured in RPMI-10% FCS (37 °C, 5% CO2) for an additional 48 h. At this time the cells were pulsed for 2 h with 1.0 µCi/well of [3H]thymidine (New England Nuclear, Boston, MA). Cells were harvested onto glass fiber mats (Skatron Instruments, Inc.,Sterling, VA), dried, and filter bound [3H]thymidine radioactivity determined (β-Plate scintillation counter, Pharmacia LKB Biotechnology, Piscataway, NJ). Inhibition of [3H]thymidine uptake was determined by comparing the mean CPM for treated samples with that of the mean CPM of the untreated control. RESULTS AND DISCUSSION

Linker Synthesis. The branched linkers were synthesized using L- or D-glutamic acid as a convenient template. The two carboxyl groups of glutamic acid were converted to hydrazides, either directly as in 2 (Scheme 2), or via an extended β-alanine spacer as in 3 (Scheme 3). The amino group of glutamic acid was extended using an amide linkage with maleimidoalkanoic acids of varying length to introduce a terminal maleimide group. The two series of linkers 2 and 3 were synthesized by parallel procedures. For the 2 series, Z-Glu was doubly activated with DCC/NHS to give the bis-succinimdyl carbonate which was directly coupled with tert-butyl carbazate to give 4. Catalytic hydrogenation gave the free amine 5. The maleimidoalkanoyl tether was attached to 5 by either of two amide coupling methods, yielding 6a6c. The unprotected bis-hydrazides 7a-7c were generated by treatment with 50% TFA in CH2Cl2. Condensation of 7a-7c with DOX in MeOH in the presence of catalytic TFA1 was monitored by RP-HPLC, which showed that the reaction progressed through a monohydrazone species to give bis-hydrazones 2a-2c. The final products were generally isolated in good yield and purity after chromatography on Sephadex LH-20 in methanol (13). Included in this series is one D-analogue, D-2b, which was synthesized by an identical procedure from Z-D-Glu. In the 3 series, the hydrazide linkage was initially installed on the β-alanine unit before attachment to Z-Glu. Thus, Z-β-Ala was activated with DCC/NHS and coupled to tert-butyl carbazate to give 8. Catalytic hydrogenation generated 9, which was coupled to Z-Glu in a manner similar to that used for 4 to generate the extended bis-hydrazide 10. Continuing the parallel sequence, 10 was elaborated to the extended bis-hydrazones 3a-3c. Our earliest attempt at the synthesis of glutamyldihydrazides made use of the attachment of a maleimidoglycine unit to 5 to give 6 (n ) 1), with the ultimate goal of extension to 2 (n ) 1). In a similar manner, 12 (n ) 1) was synthesized.2 It was felt that linkers with short tethers would be the most H2O soluble. We unexpectedly found that 6 (n ) 1) was unstable in methanol. NMR studies showed that it decomposed with a T1/2 of about 24 h at room temperature to yield a maleamate. By contrast, 12 (n ) 1) was completely stable, as were all 1 Prior experience with the synthesis of unbranched DOX hydrazones has shown TFA to be an effective catalyst in this reaction (8). Advantageously bound TFA, from the previous synthetic step, acted as catalyst in the preparation of 2a-2c. 2 Data not shown.

286 Bioconjugate Chem., Vol. 10, No. 2, 1999

King et al. Scheme 5. 2-Mercaptoethanol Adducts of 1-3

Figure 1. Relative in vitro potencies of branched and linear conjugates evaluated as a function of in vitro exposure time.

compounds with n g 2. Appparently 6 (n ) 1) is a uniquely activated electrophile toward protic solvolysis by virtue of the hydrazide groups at close proximity. Because methanol was a crucial solvent during later stages of the syntheses, we decided to abandon maleimidoglycine linkers in general. Ultimately, this proved to be inconsequential in regard to solubility, since all members of the 2 and 3 series which were synthesized were fully soluble in H2O as HCl salts. Conjugation of Linkers to Monoclonal Antibodies. We have previously shown that mild treatment of BR96 with DTT reduces a maximum of 4 interchain disulfide bonds to yield 8 thiol groups, which, upon reaction with the single-chain maleimide 1 gave immunoconjugates with MR’s equal to 8 (9). The reduction procedure is generally applicable and was used with minor modifications in this work to generate immunoconjugates of branched linkers. Thus, mAb (either BR96 or control IgG) was treated with a 7-fold excess of DTT with exclusion of O2 at 37 °C for 3 h. For syntheses e3 g, reduced mAb was purified by diafiltration in an Amicon stirred cell device. For larger scale syntheses (3-9 g), a Filtron minisette was used. In either case removal of small molecules was rapid (about 3 h for the Amicon device and 30 min for the Filtron minisette) and efficient, generating SH titers of about 8 for BR96 and 10 for IgG. Conjugation of a 1.3 molar excess of branched linkers (based on SH titer) for 30 min at 0 °C was followed by chromatography on Bio-Beads to remove unreacted linker. MR values of 14-16 DOX/mAb were obtained for the 2 series, consistent with the SH titer generated. Protein recoveries were generally good with yields in the range of 85-100%. For reasons not readily apparent, lower MR values were obtained for the 3 series than for the 2 series. Results for some representative immunoconjugates are summarized in Table 1. In our earlier work, we reported that BR96-DOX, prepared from the single-chain linker 1, was a single molecular entity with molecular weight 160000 (9). Conjugates prepared from 2 and 3 also were shown by SEC-HPLC to be monomeric, usually containing 25 >25 >25 >25 >25

a Specificity Ratio defined as the following: IC IgG-DOX/IC 50 50 BR96-DOX.

single-chain linker) at pH 5, a factor which is probably not significant in vivo. We believe that a T1/2 of 5-7 h is sufficient for efficient release of DOX within the tumor lysosome. In Vitro Potency of Linear and Branched DOX Hydrazone Conjugates. The in vitro potency of immunoconjugates prepared with different branched linkers is shown in Table 3. The conjugates evaluated in this study had molar ratios which ranged from 7.0 to 15.3, contained 2-fold increase in in vitro potency corresponding to the doubling of the DOX/mAb molar ratio is not clear at this time. However, in additional in vitro experiments (Figure 1), the relative potencies of the branched and linear conjugates were evaluated as a function of the in vitro exposure time. These studies demonstrated that the enhancement in potency of branched conjugates was seen only following exposure times of e2h (the standard exposure time for these assays). With longer exposure times, 8-24 h, the branched conjugates were approximately twice as potent as the linear 1 conjugate. These data suggest either that the branched conjugates are internalized more rapidly than the linear conjugates or that DOX is more rapidly released from the branched conjugates in lysosomes following internalization. The branched and linear chain BR96-DOX conjugates demonstrated antigen-specific cytotoxicity; however the specificity ratios of the branched conjugates were much higher than the linear conjugates. BR96-DOX conjugates were significantly more potent than IgG-DOX conjugates prepared at a similar molar ratio using the same linker.

In this work, we have introduced the novel concept of branched DOX hydrazone linkers for mAb conjugates. With a single branch point, these linkers have enabled us to successfully synthesize conjugates with mole ratios twice that of conjugates derived from similar single-chain DOX linkers. The new conjugates were generally obtained in good yield and were found to contain little or no aggregates, unreacted mAb, unreacted DOX hydrazone linkers, or free DOX. They demonstrate appropriate hydrolysis kinetics, releasing DOX rapidly at pH 5 while stable at pH 7. Conjugates derived from series 3 hydrolyze at the same rate at pH 5 as BR96-DOX, which is about twice as fast as those derived from series 2. Neither this factor nor others such as the length of the maleimide tether or the configuration of the Glu residue are reflected, though, in in vitro cytotoxicity. The BR96 branched hydrazone conjugates were more potent than the linear DOXHZN conjugate on both a DOX (4-14-fold) and a mAb (7-23-fold) basis. These data suggest that utilizing the branched hydrazone methodology offers a potential advantage in conjugate potency and provides a means to significantly reduce the amount of mAb required to achieve activity. The in vivo biology of the branched chain conjugates is the subject of a later manuscript. ACKNOWLEDGMENT

We acknowledge Michael Walker (BMS-Wallingford) and Y. Pendri (BMS-New Brunswick) for their assistance in various aspects of this work, and the BMS Analytical Chemistry Group (Ed Pack, Sadia Abid, and Tony Spears) for chromatographic assistance. Finally, we thank Drs. Dinesh Vyas and Terry Doyle for their support. Supporting Information Available: Analytical data for the compounds described in the Synthesis section. This material is available free of charge via the Internet at http://pubs.acs.org. LITERATURE CITED (1) Trail, P. A., Willner, D., and Hellstrom, K. E. (1995) Sitedirected delivery of anthracyclines for cancer therapy. Drug Dev. Res. 34, 196-209. (2) Trail, P. A., Willner, D., Lasch, S. J., Henderson, A. J., Hofstead, S. J., Casazza, A. M., Firestone, R. A., Hellstro¨m, I., and Hellstro¨m, K. E. (1993) Cure of xenografted human carcinomas by BR96-Doxorubicin immunoconjugates. Science 261, 212-215. (3) Sjogren, H. O., Isaksson, M., Willner, D., Hellstrom, I., Hellstrom, K. E., and Trail, P. A. (1997) Antitumor activity of carcinoma-reactive BR96-doxorubicin conjugate against human carcinomas in athymic mice and rats and syngeneic rat carcinomas in immunocompetent rats. Cancer Res. 57, 4530-4536. (4) Hellstro¨m, I., Garrigues, H. J., Garrigues, U., and Hellstro¨m, K. E. (1990) Highly tumor-reactive, internalizing, mouse monoclonal antibodies to Ley-related cell surface antigen. Cancer Res. 50, 2183-2190. (5) Garrigues, J., Garrigues, U., Hellstro¨m, I., and Hellstro¨m, K. E. (1993) Ley specific antibody with potent anti-tumor activity is internalized and degraded in lysosomes. Am. J. Pathol. 142, 607-622. (6) Kaneko, T., Willner, D., Monkovic, I., Knipe, J. O., Braslawsky, G. R., Greenfield, R. S., and Vyas, D. M. (1991) New hydrazone derivatives of adriamycin and their immunoconjugates - a correlation between acid stability and cytotoxicity. Bioconjugate Chem. 2, 133-141.

288 Bioconjugate Chem., Vol. 10, No. 2, 1999 (7) Braslawsky, G. R., Kadow, K. F., Knipe, J., McGoff, K., Edson, M., Kaneko, T., and Greenfield, R. S. (1991) Adriamycin(hydrazone)-antibody conjugates require internalization and intracellular acid hydrolysis for antitumor activity. Cancer Immunol. Immunother. 33, 367. (8) Greenfield, R. S., Kaneko, T., Daues, A., Edson, M. A., Fitzgerald, K. A., Olech, L., Grattan, J. A., and Braslawsky, G. R. (1990) Evaluation in vitro of adriamycin - immunoconjugates synthesized using an acid sensitive hydrazone linker. Cancer Res. 50, 6600-6607. (9) Willner, D., Trail, P. A., Hofstead, S. J., King, H. D., Lasch, S. J., Braslawsky, G. R., Greenfield, R. S., Kaneko, T., and Firestone, R. A. (1993) (6-Maleimidocaproyl)hydrazone of doxorubicin - a new derivative for the preparation of immunoconjugates of doxorubicin. Bioconjugate Chem. 4, 521-527.

King et al. (10) Trail, P. A., Willner, D., Lasch, S. J., Henderson, A. J., Greenfield, R. S., King, D., Zoeckler, M. E., and Braslawsky, G. R. (1992) Antigen specific activity of carcinoma reactive BR64-adriamycin conjugates evaluated in vitro and in human tumor xenograft models. Cancer Res. 52, 5693-5700. (11) Shih, L. B., Goldenberg, D. M., Xuan, H., Lu, H., Sharkey, R. M., and Hall, T. C. (1991) Anthracycline immunoconjugates prepared by a site specific linkage via an amino-dextran intermediate carrier. Cancer Res. 51, 4192-4198. (12) Riddles, P. W., Blakeley, R. L., and Zerner, B. (1979) Ellman’s reagent: 5,5′-Dithiobis(2-nitrobenzoic acid)-A reexamination. Anal. Biochem. 94, 75. (13) Henry, D. W., and Tong, G. L. (1978) Bis-hydrazones of daunomycin and adriamycin. U.S. Patent 4112217.

BC980100I