Bis(Hydroxamamide)-Based Bifunctional Chelating Agent for 99mTc

In addition, some chelating agents require harsh 99mTc complexation ..... High radiochemical yields were also registered at lower antibody concentrati...
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Bioconjugate Chem. 1999, 10, 9−17

9

ARTICLES Bis(Hydroxamamide)-Based Bifunctional Chelating Agent for Labeling of Polypeptides

99mTc

Le-Cun Xu,† Morio Nakayama,*,† Kumiko Harada,† Akihiko Kuniyasu,† Hitoshi Nakayama,† Seiji Tomiguchi,‡ Akihiro Kojima,‡ Mutsumasa Takahashi,‡ Masahiro Ono,§ Yasushi Arano,§ Hideo Saji,§ Zhengsheng Yao,| Harumi Sakahara,| Junji Konishi,| and Yoshitaka Imagawa⊥ Faculty of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-Honmachi, Kumamoto 862, Japan, Department of Radiology, Kumamoto University School of Medicine, Honjo, Kumamoto 860, Japan, Department of Patho-Functional Bioanalysis, Graduate School of Pharmaceutical Sciences, Department of Nuclear Medicine and Diagnostic Imaging, Graduate School of Medicine, Kyoto University, Yoshida-Shimoadachi-cho, Sakyo-ku, Kyoto 606-01, Japan, and The Chemo-Sero Therapeutic Research Institute, Kyokushi-mura, Kikuchi-gun, Kumamoto 869-12, Japan. Received February 25, 1998; Revised Manuscript Received August 10, 1998

To develop chelating molecules that provide 99mTc-labeled polypeptides of high in vivo stability and high specific activities under mild reaction conditions, an asymmetrical bis(benzohydroxamamide) compound with an amine group, 4′-aminomethyl-N,N′-trimethylenedibenzohydroxamamide [NH2C3(BHam)2], was designed and synthesized. The amine residue of NH2-C3(BHam)2 was converted to a maleimide group by reaction with N-succinimidyl-6-maleimidohexanoate, and the conjugation product was coupled to thiol groups of a monoclonal antibody against osteogenic sarcoma (OST7, IgG1) pretreated with 2-iminothiolane to prepare C3(BHam)2-OST7. 99mTc radiolabeling of C3(BHam)2OST7 was performed by the exchange reaction with [99mTc]glucoheptonate. [99mTc]C3(BHam)2-OST7 was further characterized using directly radioiodinated OST7 ([125I]OST7) and [111In]labeled OST7 with 1-[4-[(5-maleimidopentyl)amidobenzyl]ethylenediamine-N,N,N′N′-tetraacetic acid (EMCS-BzEDTA) as references. [99mTc]C3(BHam)2-OST7 was obtained with radiochemical yields of over 94% at protein concentrations as low as 0.2 mg/mL at room temperature for 1 h. [99mTc]C3(BHam)2-OST7 remained stable after incubation in freshly prepared murine plasma and in the presence of cysteine. Similar binding affinities to tumor cells were observed between [99mTc]C3(BHam)2-OST7 and [125I]OST7. When injected into normal mice, [99mTc]C3(BHam)2-OST7 exhibited radioactivity levels in the blood similar to [111In]-EMCS-Bz-EDTA-OST7 up to 24 h postinjection with significantly faster elimination rate of the radioactivity from the liver. In nude mice bearing osteogenic sarcoma, no significant differences were observed in the radioactivity levels in the blood and the tumor between [99mTc]C3(BHam)2-OST7 and [125I]OST7 at 24 h postinjection. These findings indicated that C3(BHam)2 provided 99mTc chelate of high stability at low concentrations even when conjugated to an intact antibody. Such characteristics render bis(hydroxamamide) compounds useful as chelating molecules for preparation of 99mTc-labeled polypeptides.

INTRODUCTION

Radiopharmaceuticals derived from low molecular weight polypeptides such as single-chain Fv fragment, octreotide, and chemotactic peptide have attracted attention for imaging of the sites of tumors, infection, and thrombosis (1-3). The rapid pharmacokinetic properties of these molecules enable target visualization at early postinjection intervals using radionuclides with short half-lives. 99mTc is an ideal radionuclide for scintigraphic * To whom correspondence should be addressed. Phone: 8196-371-4358.Fax: 81-96-372-7182.E-mail: [email protected]. † Kumamoto University. ‡ Kumamoto University School of Medicine. § Graduate School of Pharmaceutical Sciences. | Kyoto University. ⊥ The Chemo-Sero Therapeutic Research Institute.

imaging applications due to its excellent physical properties, low cost, and ready availability (4). Since most polypeptides do not possess binding sites to form 99mTc chelates of high in vivo stability, appropriate chelating molecules are incorporated into polypeptide molecules to prepare 99mTc-labeled peptides for in vivo applications. Previous efforts directed toward the conjugation of 99mTc to polypeptides include the use of tetradentate ligands with N3S or N2S2 ligand systems, which contain one or two thiol groups (5-9). While the thiol groups in the molecules facilitate the formation of mononuclear 99mTc complexes with high stability, the presence of the free thiol group may restrict the conjugation reactions with polypeptides and subsequent storage of the resulting bioconjugates. Protection of a thiol group was performed to conjugate a N3S chelating molecule to an antibody fragment, which necessitated a deprotection step before

10.1021/bc980024j CCC: $18.00 © 1999 American Chemical Society Published on Web 12/22/1998

10 Bioconjugate Chem., Vol. 10, No. 1, 1999

Xu et al.

Scheme 1

99m

Tc complexation reaction (9). Exchange reactions between the free thiol groups of the chelating agents and disulfide bonds in peptides may also constitute a potential problem in the conjugation reactions. In addition, some chelating agents require harsh 99mTc complexation conditions (elevated temperatures or high pH) to prepare 99mTc chelates with high radiochemical yields (10-12). Thus, development of thiol-free chelating molecules that provide 99mTc complexes with high in vivo stability and high specific activities under mild complexation conditions would facilitate further application of 99mTc to a variety of polypeptides of interest for diagnostic nuclear medicine. Hydrazino nicotinamide (HYNIC) is a representative thiol-free chelating agent, and applicability of this chelating agent for 99mTc labeling of polypeptides has been well demonstrated (13-15). HYNIC has been reported to act as a mono- or bidentate ligand and forms 99mTc chelates of high specific activity with high radiochemical yields in the presence of appropriate co-ligands such as glucoheptonate or tricine. We initiated a program to develop new thiol-free chelating molecules with a tetradentate ligand system. Previous studies suggested that N-hydroxy-carboximidamide (hydroxamamide, abbreviated as Ham)-based tetradentate ligands, bis(Ham), form 99mTc chelates with high stability and high specific activity with high radiochemical yields (16-19). These findings prompted us to design bis(Ham) derivatives with a functional group for the conjugation with polypeptides. In the present study, an asymmetrical bis(benzohydroxamamide) compound with an amine group at the conjugation site, 4′-aminomethyl-N,N′-trimethylenedibenzohydroxamamide [NH2-C3(BHam)2], was designed and synthesized as outlined in Scheme 1. The ability of NH2-C3(BHam)2 as a chelating molecule for 99mTc-

labeled polypeptides was estimated using a monoclonal antibody (mAb) against osteogenic sarcoma (OST7, IgG1) as a model. For comparison, OST7 was labeled with radioiodine by direct iodination and with indium-111 (111In)using1-[4-[(5-maleimidopentyl)amidobenzyl]ethylenediamine-N,N,N′N′-tetraacetic acid (EMCS-Bz-EDTA) as the chelating agent. Chemical structures of each conjugate are illustrated in Figure 1. MATERIALS AND METHODS

Reagents and Chemicals. Proton nuclear magnetic resonance (1H NMR) spectra were recorded on a JNM500 (500 MHz) instrument (JEOL Ltd., Tokyo, Japan). Electron impact mass spectra (EI-MS) and fast atom bombardment mass spectra (FAB-MS) were obtained with a JMS-DX303HF mass spectrometer (JEOL Ltd.). Melting points were determined with a Yanaco Micro Melting Point Apparatus and were reported uncorrected. Na[125I]I was obtained from Daiichi- Kagaku (Tokyo) and diluted with phosphate buffer (PB; 0.1 M, pH 7.4) to 0.37-3.7 MBq (0.01-0.1 mCi)/µL before use. 111InCl3 [74 MBq (2 mCi)/mL in 0.02 N HCl] was supplied by NihonMedi-Physics (Tokyo, Japan). [99mTc]Pertechnetate (99mTcO4-) was eluted in saline solution on a daily basis from Daiichi Radioisotope Labs. generators (Chiba, Japan). 3-Phenyl-4-(3-bromopropyl)-∆2-1, 2, 4-oxadiazolin5-one (compound 6 in Scheme 1) was synthesized according to the procedures described previously (19). Both N-(maleimidocaproyloxy)succinimide (EMCS) and EMCSBz-EDTA were purchased from Dojindo Labs (Kumamoto, Japan). 2-Iminothiolane (2-IT) was obtained from Nacalai Tesque, Inc. Kyoto, Japan). Size-exclusion HPLC was performed using a TSK guard column SWXL (6.0 × 40 mm) connected to a TSK G3000SWXL (7.8 × 300 mm) column, eluted with 0.1 M PB (pH 7.0) containing 0.3 M

Hydroxamamide-Based Bifunctional Chelating Agent

Bioconjugate Chem., Vol. 10, No. 1, 1999 11

Figure 1. Chemical structures of C3(BHam)2-OST7 conjugate (A), EMCS-Bz-EDTA-OST7 conjugate (B) and (C).

sodium chloride at a flow rate of 0.5 mL/min. The eluent was monitored at 280 nm with an ultraviolet (UV) detector (Hitachi L-400 UV detector; Hitachi, Tokyo) and a well-type NaI (Tl) scintillation detector (Aloka PS-201; Aloka, Tokyo) coupled with a universal scalar (Aloka TCD 501). TLC analyses were performed with cellulose plates (Merck 5577) using 0.02 M phosphate-buffered saline (PBS, pH 7.0) as the developing solvent. Under these conditions, protein-bound radioactivity remained at the origin while 99mTcO4- and [99mTc]cysteine moved with the solvent front. Cellulose acetate electrophoresis (CAE) was run on Separax SP (Joko, Tokyo) at a constant current of 0.8 mA/cm for 30 min in 0.072 M veronal buffer (pH 8.6). The migration distance of the antibody was determined by Ponceau 3R staining. To facilitate collection of urine and feces after administration of radiolabeled antibodies, mice were housed in metabolic cages (metabolica, MM type; Sugiyama-Gen Iriki Co. Ltd., Tokyo). Tumor and mAb. KT005-cloned human osteosarcoma was maintained by serial subcutaneous transplantation in athymic nude mice. Female BALB/c athymic mice were implanted s.c. with 1-2 mm cubed pieces of KT005. Tumors of 0.2-0.5 g in weight at ca. 2 weeks postplantation were used in vivo study. Single-cell suspensions from xenografted tumors were used for the in vitro study (20,21). The mAb against osteogenic sarcoma (OST7, IgG1) was generated by a standard hybridoma technique, as described previously (22). Synthesis of p-[N-(tert-Butoxycarbonyl)aminomethyl]benzonitrile (2). To a solution of p-(aminomethyl)benzonitrile (1, 10 g, 0.075 mol) in 60 mL N,N′dimethyformamide (DMF) was gradually added a solution of (Boc)2O (17.4 g, 0.080 mol) in 50 mL of DMF, and the mixture was stirred at room temperature for 1 h. After removing the solvent in vacuo (30 mmHg), water (100 mL) was added to produce a white precipitate. The precipitate was collected and recrystallized from methanol-water (5:4) to produce compound 2 (17.5 g) in 99% yield. Mp 108-109 °C. 1H NMR (DMSO-d6): δ 7.479 (s, 1H, NH), 7.399-7.776 (m, 4H, aromatic H), 4.186 (s, 2H, NCH2), 1.381 (s, 9H, (CH3)3C). EI-MS calculated for C13H17N2O2 (M+): m/z 232. Found: 232. Anal. Calcd for C13H16N2O2: C, 67.22; H, 6.94; N, 12.06. Found: C, 67.46; H, 7.15; N, 11.85.

125I-labeled

OST7

Synthesis of p-[N-(tert-Butoxycarbonyl)aminomethyl]benzohydroxamamide (3). A solution of free hydroxylamine was prepared by treating 13.8 g (0.20 mol) of hydroxylamine hydrochloride with 18 g (0.20 mol) of sodium bicarbonate in 100 mL of water. Compound 2 (36.8 g, 0.15 mol) in 100 mL of methanol was then added to the solution of free hydroxylamine, and the mixture was stirred at 60 °C for 8 h. After removing the solvent in vacuo, 100 mL of water was added to produce a white precipitate. Compound 3 was obtained after recrystallization of the precipitate from ethanol-water (1:1) with 94% (37.3 g) yield. Mp 152-153 °C. 1H NMR (DMSOd6): δ 9.550 (s, 1H, OH), 7.364 (s, 1H, NHCH2), 7.2037.608 (m, 4H, aromatic H), 5.737 (s, 2H, NH2), 4.120 (d, 2H, NHCH2), 1.386 (s, 9H, (CH3)3C). EI-MS calculated for C13H20N3O3 (M+): m/z 265. Found: 265. Anal. Calcd for C13H19N3O3: C, 58.85; H, 7.22; N, 15.84. Found: C, 59.08; H, 7.17; N, 15.70. Synthesis of p-[N-(tert-Butoxycarbonyl)aminomethyl]benzamamide O-Ethoxycarbonyloxime (4). To a chilled solution of compound 3 (37 g, 0.14 mol) in 350 mL of dry acetone in an ice bath was slowly added 15.2 g (0.14 mol) of ethyl chlorocarbonate while maintaining the reaction temperature. A white precipitate of hydroxamamide hydrochloride was produced. While maintaining the reaction temperature at 0-4 °C, a solution of NaOH (5.7 g, 0.14 mol) in 100 mL of water was then gradually added. After stirring for 1 h at room temperature, the solvent was removed in vacuo. The solid residue was recrystallized from ethanol-water (1:1) to produce 37 g of compound 4 as white crystals (78%). Mp 109-110 °C. 1H NMR (DMSO-d6): δ 7.413 (s, 1H, NHCH2), 7.270-7.631 (m, 4H, aromatic H), 6.749 (s, 2H, NH2), 4.152 (d, 2H, NHCH2), 4.175 (q, 2H, J ) 7.32 Hz, OCH2CH3), 1.251 (t, 3H, J ) 7.32 Hz, OCH2CH3), 1.386 (s, 9H, (CH3)3C). EI-MS calculated for C16H24N3O5 (M+): m/z 337. Found: 337. Anal. Calcd for C16H23N3O5: C, 56.96; H, 6.87; N, 12.45. Found: C, 57.21; H, 6.96; N, 12.54. Synthesis of 3-[p-(N-(tert-Butoxycarbonyl)aminomethyl)phenyl]-∆2-1,2,4-oxadiazolin-5-one (5). Compound 4 (36 g, 0.11 mol) was added to a solution of NaOH (17 g) in 300 mL of water, and the mixture was heated in a boiling water bath for 10 min. After the solution

12 Bioconjugate Chem., Vol. 10, No. 1, 1999

became clear, the reaction mixture was cooled in an ice bath and acidified with acetic acid to produce a while precipitate. The precipitate was collected and recrystallized from ethanol-water (5:6) to yield 28 g of compound 5 as white crystal (87%). Mp 176-177 °C. 1H NMR (DMSO-d6): δ 7.441 (s, 1H, NHCH2), 7.397-7.753 (m, 4H, aromatic H), 4.186 (d, 2H, NHCH2), 1.385 (s, 9H, (CH3)3C). EI-MS calculated for C14H18N3O4 (M+): m/z 291. Found: 291. Anal. Calcd. for C14H17N3O4: C, 57.72; H, 5.88; N, 14.42. Found: C, 57.61; H, 5.85; N, 14.28. Synthesis of 3-[p-(N-(tert-Butoxycarbonyl)aminomethyl)phenyl]-3′-phenyl-4,4′-trimethylenedi-∆21, 2, 4-oxadiazolin-5-one (7). To a solution of compound 5 (15.5 g, 0.053 mol) in methanol (40 mL) was added a solution of KOH (3.0 g, 0.053 mol) in 50 mL of methanol. After stirring for 10 min, the solvent was removed in vacuo. The residue was dissolved in 80 mL of dry DMF, and 13.5 g (0.047 mol) of 3-phenyl-4-(3-bromopropyl)-∆21, 2, 4-oxadiazolin-5-one (19) was added. After 3 days of stirring at 50 °C, the precipitates were filtered off and the filtrate was evaporated in vacuo. Chloroform (30 mL) was added to the residue, and the organic phase was washed sequentially with 1.0 M NaOH and water. After drying over anhydrous MgSO4, the solvent was removed in vacuo. The solid residue was crystallized from ethanol to give 14.9 g of compound 7 (57%) as white crystals. Mp 166-167 °C. 1H NMR (DMSO-d6): δ 7.415-7.699 (m, 9H, aromatic H), 4.228-4.241 (d, 2H, NH2CH2), 3.557-3.584 (m, 4H, CH2CH2CH2), 1.746-1.774 (m, 2H, CH2CH2CH2), 1.411 [s, 9H, (CH3)3C]. FAB-MS calculated for C25H28N5O6 (MH+): m/z 493. Found: 493. Anal. Calcd for C25H27N5O6: C, 60.84; H, 5.51; N, 14.19. Found: C, 60.49; H, 5.56; N, 13.88. Synthesis of 3-[p-(Aminomethyl)phenyl]-3′-phenyl-4,4′-trimethylenedi-∆2-1,2,4-oxadiazolin-5-one (8). Compound 7 (2.47 g, 0.005 mol) was dissolved in a mixed solution of trifluoroacetic acid (25 mL) and anisole (25 mL) at 0 °C. After stirring at room temperature for 1 h, the solvent was removed in vacuo. The residue was dissolved in water, and 1 N NaOH was added to bring the solution pH to ca. 9-10. A white precipitate was obtained after chilling the solution in an ice bath. Recrystallization of the precipitate from ethanol produced compound 8 (1.5 g, 74%). Mp 157-158 °C. 1H NMR (DMSO-d6): 7.485-7.704 (m, 9H, aromatic H), 3.5513.584 (m, 4H, CH2CH2CH2), 3.828 (s, 2H, NH2CH2), 1.724-1.1.783 (m, 2H, CH2CH2CH2). FAB-MS calculated for C20H20N5O4 (MH+): m/z 393. Found: 393. Anal. Calcd. for C20H19N5O4‚1/2H2O: C, 59.7; H, 5.01; N, 17.4. Found: C, 59.96; H, 4.851; N, 17.17. Synthesis of NH2-C3(BHam)2. Compound 8 (1.0 g, 2.5 mmol) was added to 20 mL of 5% NaOH, and the mixture was heated with stirring at 90 °C for 10 min. After cooling to 0-4 °C in an ice bath, the reaction mixture was neutralized with 1 M HCl. A white precipitate was obtained after cooling the reaction solution on ice for several hours. The precipitate was collected and crystallized from ethanol to give 0.6 g (1.8 mmol) of NH2C3(BHam)2 as white crystals (72%). Mp 179-180 °C. 1H NMR (DMSO-d6): δ 9.705 (s, 2H, OH), 8.213 (s, 2H, NH2CH2), 7.332-7.473 (m, 9H, aromatic H), 5.642-5.694 (m, 2H, HNCH2CH2CH2NH), 4.037 (s, 2H, NH2CH2), 2.8582.897 (m, 4H, HNCH2CH2CH2NH), 1.366-1.392 (m, 2H, HNCH2CH2CH2NH). FAB-MS calcd for C18H23N5O2 (MH+): m/z 341. Found: 341. Anal. Calcd for C18H23N5O2: C, 63.32; H, 6.79; N, 20.51. Found: C, 63.42; H, 7.02; N, 20.28. Preparation of C3(BHam)2-OST7 Conjugate. The conjugation reaction of NH2-C3(BHam)2 with OST7 was

Xu et al.

performed according to the procedure described previously (23) with slight modifications as follows. To 0.5 mL of a solution of OST7 (9 mg/mL) in well-degassed 0.16 M borate buffer (pH 8.0) containing 2 mM ethylenediaminetetraacetic acid (EDTA) was added 25 µL of 2-IT (2 mg/ mL) in the same buffer. After gentle agitation at room temperature for 1 h, excess 2-IT was removed by the centrifugation through gel-filtration columns packed with Sephadex G-50 (Pharmacia Biotech, Tokyo) equilibrated with 0.1 M PBS (pH 5.5) containing 2 mM EDTA. EMCS in DMF (50 µL, 40 mM) was added to an equal volume of NH2-C3(BHam)2 (80 mM) in the same solvent. After stirring the reaction mixture for 30 min at room temperature, 20 µL of this solution was slowly added to the freshly thiolated OST7 solution (0.5 mL) and the reaction mixture was stirred gently at room temperature for 2 h. The solution was further agitated gently for 30 min after addition of iodoacetamide (20 µL, 10 mg/mL) to alkylate the unreacted thiol groups. The C3(BHam)2-OST7 conjugate was separated from unreacted small molecules using Sephadex G-50 column chromatography (1.5 × 37 cm) equilibrated and eluted with PBS (pH 7.0). The number of C3(BHam)2 chelates introduced per molecule of OST7 was estimated by measuring the number of thiol groups of OST7 before and after addition of the reaction mixture of EMCS and NH2-C3(BHam)2 using 2,2′-dithiopyridine (24). C3(BHam)2-OST7 was characterized by SDS-PAGE using 4% stacking gels and 8% running gels according to the method of Laemmli under nonreducing conditions (25). After mixing with 4% SDS, the samples were immersed for 5 min in a boiling water bath and loaded on the gels at a concentration of 4.0 µg of protein per lane. Electrophoresis was performed with a current of 20 mA per gel until the bromophenol blue marker reached the bottom of the gel. After running, gels were stained with 0.02% Coomassie brilliant blue (CBB) R250 in 25% methanol/10% acetic acid, and subsequently destained by equilibrating with 5% acetic acid/2% glycerol. 99mTc Labeling of C (BHam) -OST7. C (BHam) 3 2 3 2 OST7 was labeled with 99mTc by reaction with [99mTc]glucoheptonate. A freshly collected solution (3-4 mL) of 99m TcO4- [1 mCi (37 MBq)/mL] was added to a vial containing lyophilized glucoheptonate (4 mg) and SnCl2 (60 µg) under a nitrogen atmosphere and allowed to stand for 15 min at room temperature. Aliquots of this solution (200 µL) were added to the C3(BHam)2-OST7 solution (200 µL) of varying protein concentrations and incubated for 1 h at room temperature. Radiochemical purities of [99mTc]C3(BHam)2-OST7 were determined by TLC, CAE, size-exclusion HPLC, and SDS-PAGE. Autoradiography of the dried PAGE gels was carried out by placing them on X-ray film (Fuji Photo Film Inc., Tokyo, Japan) for 12 h. Stability of 99mTc-Labeled C3(BHam)2-OST7. [99mTc]C3(BHam)2-OST7 (0.2 mg/mL; 20 µL) was added to a tube containing 200 µL of freshly prepared murine plasma to a final protein concentration of 1.3 × 10-7 M. After incubating the solution at 37 °C for 6 and 18 h, the mixture was analyzed by CAE and TLC. Stabilities of [99mTc]C3(BHam)2-OST7 were also estimated in the presence of cysteine. A 10 µL aliquot of 1.2 × 10-6 M [99mTc]C3(BHam)2-OST7 in 0.1 M PBS (pH 7.0) was mixed with 10 µL of cysteine to make the final concentrations of cysteine in the range of 3.6 × 10-6 to 3.6 × 10-4 M. The mixture was incubated at 37 °C for 1 and 6 h and analyzed by TLC using 0.005 M PB (pH 7) as the eluent. In this TLC system, [99mTc]cysteine showed an

Hydroxamamide-Based Bifunctional Chelating Agent

Rf value above 0.8, while [99mTc]C3(BHam)2-OST7 remained at the origin. Preparation of [111In]EMCS-Bz-EDTA-OST7 and [125I]OST7. Conjugation of EMCS-Bz-EDTA to OST7 was performed by maleimide-thiol chemistry using 2-IT, and the conjugate was labeled with 111In, as described previously (24, 26). The number of chelating agents attached per molecule of OST7 was 2.2 as determined by calculating the number of thiol groups per molecule of OST7 before and after conjugation with EMCS-BzEDTA. Radiochemical purity of [111In]EMCS-Bz-EDTAOST7 was over 92% as determined by CAE analysis. Direct radioiodination of OST7 was performed by the chloramine-T method (27). To 100 µL of OST7 (0.25 mg/ mL) in 0.3 M phosphate buffer (pH 7.3) was added 2 µL of Na[125I]I and 12.5 µL of chloramine-T solution (0.1 mg/ mL) in the same buffer. After 10 of min incubation at room temperature, the reaction was terminated by addition of 3 µL of aqueous sodium bisulfite (0.7 mg/mL). [125I]OST7 was purified by the centrifuged gel-filtration column method using Sephadex G-50 as described above. Radiochemical purity of the radioiodinated OST7 was over 97% as determined by CAE analysis. Immunoreactivity Measurement. The immunoreactivities of [99mTc]C3(BHam)2-OST7 and [125I]OST7 were determined as described previously (20, 21, 23). Singlecell suspensions from xenografted tumors [(1 × 104)-(1 × 107)] in 100 µL of Dulbecco’s PBS were incubated with 100 µL of radiolabeled OST7s in microcentrifugation tubes (5.7 × 46 mm) for 2 h at 4 °C. After centrifugation at 10000g, the supernatant was aspirated off and the radioactivity was determined using an Aloka ARC-2000 auto-well γ system (Aloka, Tokyo). In Vivo Studies. An aliquot of 120 µL of [99mTc]C3(BHam)2-OST7 [0.5 mCi (18.5 MBq)/0.2 mg/0.4 mL] or 50 µL of [111In]EMCS-Bz-EDTA-OST7 [10 µCi (370 KBq)/40 µg/50 µL] was added to 3 mL of unmodified OST7 (0.2 mg/mL) in 0.1 M PBS (pH 7.0). The biodistribution of radioactivity after intravenous administration of each radiolabeled OST7 was monitored at 1, 3, 6, and 24 h postinjection into 5-week-old male ddY mice weighing 20-25 g (28). Groups of five mice each receiving ca. 20 µg of OST7 were used for the experiment (20, 21, 23, 29). Organs or tissues of interest were removed and weighed, and their radioactivities were determined using an Aloka ARC-2000 auto-well γ system. An aliquot of 40 µL of [99mTc]C3(BHam)2-OST7 [0.5 mCi (18.5 MBq)/0.2 mg/0.4 mL] and 30 µL of [125I]OST7 [1 µCi (37 KBq)/2.5 µg/10 µL] were added to 1 mL of unmodified OST7 (0.2 mg/mL). Athymic mice bearing osteogenic sarcoma were treated intravenously with 100 µL of the mixture. At 24 h postinjection, the animals were sacrificed and the organs or tissues of interest were removed and weighed, and the radioactivity in each organ or tissue was measured immediately for 99mTc and 3 days later for 125I using an Aloka ARC-2000 auto-well γ system. Statistical Analyses. Data are expressed as means (standard deviation) where appropriate. Results were statistically analyzed using unpaired Mann-Whitney test for in vivo studies. Differences were considered significant when p value was less than 0.05. RESULTS

Synthesis of NH2-C3(BHam)2. The asymmetrical bis(Ham) compound NH2-C3(BHam)2 was synthesized according to the synthetic procedure reported previously for symmetrical bis(Ham) compounds (19). After protect-

Bioconjugate Chem., Vol. 10, No. 1, 1999 13 Table 1. Radiochemical Yields of [99mTc]C3(BHam)2-OST7 at Different Antibody Concentrations concentration of C3(BHam)2OST7 conjugate (mg/mL)

labeling yieldsa (%)

2.0 1.0 0.5 0.2 unmodified OST7 (0.2 mg/mL)

98.1 ( 1.7 96.0 ( 1.5 94.5 ( 1.9 94.3 ( 1.7