Assessment of Radiochemical Design of Antibodies Using an Ester

Reduction of radioactivity levels in nontarget tissues such as the liver and kidney constitutes a problem to be resolved in diagnostic and therapeutic...
3 downloads 0 Views 427KB Size
Download PDF
Bioconjugate Chem. 1996, 7, 628−637

628

Assessment of Radiochemical Design of Antibodies Using an Ester Bond as the Metabolizable Linkage: Evaluation of Maleimidoethyl 3-(Tri-n-butylstannyl)hippurate as a Radioiodination Reagent of Antibodies for Diagnostic and Therapeutic Applications Yasushi Arano,*,† Kouji Wakisaka,† Yoshiro Ohmono,§ Takashi Uezono,† Hiromichi Akizawa,† Morio Nakayama,# Harumi Sakahara,‡ Chiaki Tanaka,§ Junji Konishi,‡ and Akira Yokoyama† Faculty of Pharmaceutical Sciences, Department of Radiopharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-01, Japan, Faculty of Medicine, Department of Nuclear Medicine, Kyoto University, Sakyo-ku, Kyoto 606-01, Japan, Department of Radiopharmaceutical Sciences, Osaka University of Pharmaceutical Sciences, Matsubara 567, Japan, and Faculty of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-Honmachi, Kumamoto 862, Japan. Received September 5, 1995X

Reduction of radioactivity levels in nontarget tissues such as the liver and kidney constitutes a problem to be resolved in diagnostic and therapeutic applications of radiolabeled monoclonal antibodies (mAbs). A new radioiodination reagent with an ester bond to liberate m-iodohippuric acid from covalently conjugated proteins, maleimidoethyl 3-(tri-n-butylstannyl)hippurate (MIH), was recently developed. MIH liberated m-iodohippuric acid from galactosylneoglycoalbumin in murine liver, and the radiometabolite was rapidly eliminated from the liver into urine as an intact structure. In this study, intact IgG and Fab fragment of a mAb against osteogenic sarcoma were radioiodinated with MIH to further assess the applicability of MIH to radioimmunoimaging and therapy. For comparison, a mAb radioiodinated with N-succinimidyl iodobenzoate (SIB) and indium-111 (111In)-labeled mAbs with diethylenetriaminepentaacetic dianhydride (cDTPA) or 1-[4-[(5-maleimidopentyl)amino]benzyl]ethylenediaminetetraacetic acid (EMCS-Bz-EDTA) were used. Size-exclusion HPLC analysis and cell binding assays indicated the preservation of both structure and antigen binding affinity of radioiodinated MIH-OST7 (IgG). In biodistribution studies in mice, [125I]MIH-OST7 (IgG) showed faster systemic clearance of radioactivity after 24 h postinjection than did [131I]SIB- and [111In]EMCS-BzEDTA-OST7 (IgG). [125I]MIH-OST7 (IgG) also exhibited much lower radioactivity levels in nontarget tissues such as the liver and kidney, with higher radioactivity levels in the blood up to 72 h postinjection when compared with [111In]cDTPA-OST7 (IgG). Radioactivity excreted from the mice was found in the urine as m-iodohippuric acid, following administration of [125I]MIH-OST7 (IgG). In athymic mice bearing osteogenic sarcoma, [131I]MIH-OST7 (IgG) indicated higher tumor-to-nontarget ratios of radioactivity at both 24 and 48 h postinjection than [125I]SIB-OST7 (IgG). Although both radioiodinated OST7s showed similar radioactivity levels in the target at 24 h postinjection, a small but significant decrease in the target radioactivity level was observed with [131I]MIH-OST7 (IgG) at 48 h postinjection. In addition, [131I]MIH-OST7 (Fab) showed very rapid cleavage of the ester bond both in vivo and in vitro. These findings indicated that while MIH may be a useful reagent for radioimmunoimaging using IgG mAb, its application to smaller molecular weight mAbs and radioimmunotherapy would be hindered due to the labile characteristics of the ester bond in plasma. Thus, while the present study reinforced the usefulness of metabolizable linkages for reducing nontarget radioactivity levels, a development of plasma-stable metabolizable linkages is also warranted for radioimmunotherapy and for smaller molecular weight polypeptides.

INTRODUCTION

Recent advances in genetic and protein engineering have provided monoclonal antibodies (mAbs) with reduced or no immunogenicity (e.g. chimeric or humanized mAbs) or more favorable distribution characteristics to target tissues (e.g. single-chain Fv fragment) (1-3). Radiochemists have developed various reagents that allow stable attachment of radiolabels to mAbs in vivo * Author to whom correspondence should be addressed [telephone (075)753-4556; fax (075) 753-4568; e-mail arano@ pharmsun.pharm.kyoto-u.ac.jp]. † Department of Radiopharmaceutical Sciences, Kyoto University. § Osaka University of Pharmaceutical Sciences. # Kumamoto University. ‡ Department of Nuclear Medicine. X Abstract published in Advance ACS Abstracts, October 15, 1996.

S1043-1802(96)00058-4 CCC: $12.00

(4-6). Efforts have also been made to increase the target radioactivity levels by changing the administration routes, by hyperthermia, or by using biological response modifiers with various mechanisms of action (7). These approaches would eliminate problems associated with the clinical application of radiolabeled mAbs. However, localization of radioactivity in nontarget tissues still constitutes a problem to be resolved, and the development of radiochemistry of mAbs that can decrease the undesirable radioactivity localization is highly warranted for both diagnostic and therapeutic applications of the radiopharmaceuticals. Furthermore, the development of such chemistry would also be useful for radiolabeling of smaller molecular weight peptides such as octreotide and chemotactic peptides. Among the various strategies to decrease the nontarget localization of radioactivity following administration of radiolabeled mAbs, use of a “metabolizable linkage” constitutes one attractive approach (8-14). This radio© 1996 American Chemical Society

Metabolizable Ester Bond in Radioimmunoconjugates

chemical design of mAbs allows excretion of the radioactivity accumulated in nontarget tissues into the urine through the introduction of a cleavable linkage between the mAb and the radiolabel. The rationale behind the radiochemical design of mAbs is supported by recent metabolic studies of radiolabeled polypeptides, indicating that radiometabolites generated after lysosomal proteolysis of polypeptides play an important role in the radioactivity levels in the liver (15-18). These studies also indicated the importance of the chemical and biological properties of radiometabolites in the radiochemical design of mAbs using metabolizable linkages; even if a suitable metabolizable linkage is placed between the mAb and radiolabel, little or no improvement in the radioactivity levels in the nontarget tissues would be achieved if the radiometabolite is retained in or only slowly excreted from the lysosomal compartment of nontarget tissues. Therefore, metabolizable linkages should be designed to liberate radiometabolites with rapid urinary excretion from covalently conjugated mAbs after lysosomal proteolysis in nontarget tissues. Recently, we developed a novel radioiodination reagent with an ester bond to liberate m-iodohippuric acid, maleimidoethyl 3-(tri-n-butylstannyl)hippurate (MIH) (19). Preliminary studies using galactosylneoglycoalbumin (NGA) as a model protein indicated that MIH liberates m-iodohippuric acid as the major radiometabolite after lysosomal proteolysis in hepatic parenchymal cells, and the metabolite was excreted rapidly from the hepatocytes into the urine intact. Thus, MIH is a radioiodination reagent that liberates a radiometabolite of known chemical and biological properties after cleavage of the ester bond from the covalently conjugated protein in hepatic parenchymal cells, where nonspecific localization of radioactivity is observed in mice when radiolabeled mAbs are administered (20, 21). Such chemical and biological characteristics make MIH a potentially useful reagent for radioiodination of mAbs for diagnostic and therapeutic purposes. Furthermore, since meta-iodohippuric acid possesses almost ideal chemical and biological characteristics as a radiometabolite, use of MIH would provide insight for estimation of the usefulness of ester bonds as metabolizable linkages. To assess the applicability of MIH to radioimmunoimaging and therapy, an intact IgG (IgG1) and Fab fragment of a mAb against osteogenic sarcoma (OST7) were radioiodinated using MIH. Stabilities of the ester bonds in MIH-labeled OST7s were estimated. Biodistributions of radioactivity of MIH-labeled OST7 (IgG) were investigated in normal and athymic mice bearing osteogenic sarcoma. Radioiodinated OST7 (IgG and Fab) with N-succinimidyl 3-iodobenzoate (SIB) and indium-111 (111In)-labeled OST7s (IgG) with diethylenetriaminepentaacetic dianhydride (cDTPA) or 1-[4-[(5-maleimidopentyl)amino]benzyl]ethylenediaminetetraacetic acid (EMCSBz-EDTA) as bifunctional chelating agents (BCAs) were used as references. Chemical structures of each conjugate are illustrated in Figure 1. SIB generates radioiodinated mAbs with high stability against in vivo deiodination and is superior to direct radioiodination of mAbs for both diagnostic and therapeutic purposes (6). The benzyl-EDTA chelating site in EMCS-Bz-EDTA forms an 111 In chelate of high in vivo stability (16, 22) and the chelating agent can be attached to OST7 (IgG) by conjugation procedures similar to those used for radioiodinated MIH. cDTPA has been a widely used BCA for 111 In radiolabeling of mAbs. Furthermore, radiometabolites generated from the respective reagent after lysosomal proteolysis in murine hepatocytes have been well characterized in previous studies (16-19, 23, 24). The

Bioconjugate Chem., Vol. 7, No. 6, 1996 629

Figure 1. Chemical structures of MIH-labeled OST7 (IgG) (A), 1-[4-[(5-maleimidopentyl)amino]benzyl]ethylenediaminetetraacetic acid (EMCS-Bz-EDTA)-conjugated OST7 (IgG) (B), Nsuccinimidyl 3-iodobenzoate (SIB)-labeled OST7 (IgG) (C), and cDTPA-conjugated OST7 (D). While MIH and EMCS-Bz-EDTA were attached to OST7 by similar conjugation procedures, cDTPA and SIB were attached to amine residues of OST7. MIH and SIB possess a m-iodobenzoyl group to stabilize iodine against in vivo deiodination.

persistent retention of radiometabolites in hepatocytes derived from cDTPA-111In would be useful for estimation of the role played by the ester bond in MIH. The radiochemical design of mAbs using an ester bond as the metabolizable linkage was also evaluated for diagnostic and therapeutic applications in nuclear medicine. MATERIALS AND METHODS

Reagents and Chemicals. Na[131I]I and Na[125I]I were obtained from Daiichi Radioisotopes Laboratories (Tokyo, Japan) and Daiichi Kagaku (Tokyo), respectively, and were diluted with phosphate buffer (PB; 0.1 M, pH 7.4) to 7.4 MBq (0.2 mCi)/µL and 3.7 MBq (0.1 mCi)/µL, respectively. 111InCl3 (74 MBq (2 mCi)/mL in 0.02 N HCl) was supplied by Nihon Medi-Physics (Takarazuka, Japan). MIH was synthesized as described previously (19). N-Succinimidyl 3-(tri-n-butylstannyl)benzoate (ATE) was prepared according to the method of Zalutsky et al. (6). Both cDTPA and EMCS-Bz-EDTA were purchased from Dojindo Laboratories (Kumamoto, Japan). Size-exclusion HPLC and reversed-phase (RP) HPLC were performed using Cosmosil Diol-300 (7.5 × 600 mm, Nacalai Tesque, Kyoto, Japan) and Cosmosil 5C18-AR (4.6 × 250 mm, Nacalai Tesque), respectively. Size-exclusion HPLC and RP-HPLC columns were equilibrated and eluted with 0.1 M phosphate buffer (PB; pH 6.8) and with a mixture of 0.1% aqueous phosphoric acid-acetonitrile (7:3), respectively, at a flow rate of 1 mL/min. Other reagents were of reagent grade and used as received. Tumor and mAbs. KT005-cloned human osteogenic sarcoma was maintained by serial subcutaneous transplantation in athymic mice. Pieces of tumor tissue (1-2 mm3; 0.3-0.5 g) at ca. 2 weeks postplantation were used for the in vivo study. Single-cell suspensions from xenografted tumors were used for the in vitro study (25, 26). The mAb against osteogenic sarcoma (OST7, IgG1), generated by standard hybridoma technology, was purified by ammonium sulfate precipitation with subsequent protein A affinity chromatography (Pharmacia Biotech

630 Bioconjugate Chem., Vol. 7, No. 6, 1996

Co. Ltd., Tokyo) (27). The Fab fragment of OST7 was prepared according to the standard procedure (28). Preparation of [125/131I]MIH-Labeled OST7s (IgG and Fab). Radioiodination of MIH with Na[131I]I was performed in the presence of N-chlorosuccinimide (NCS) used as an oxidant, as described previously (19). Briefly, a 22.0 µL solution of MIH in methanol containing 1% acetic acid (0.56 mg/mL) was mixed with 6.04 µL of NCS in methanol (0.5 mg/mL) in a sealed vial, followed by an addition of Na[131I]I (3 µL). After incubation of the reaction mixture for 45 min at room temperature, the reaction was quenched with aqueous sodium bisulfite (2.92 µL, 0.72 mg/mL). The radiochemical yields of [131I]MIH were determined by TLC (Merck Art. 5553) developed with a mixture of chloroform-ether (1:1). The solvent was removed under a flow of N2 prior to subsequent conjugation reactions with OST7s. Radioiodination of MIH with Na[125I]I was performed as described above except using Na[125I]I in place of Na[131I]I. Conjugation of [125/131I]MIH with OST7 (IgG) was performed by reducing the disulfide bonds of OST7, as described previously (8). Briefly, OST7 (IgG) (300 µL, 2.5 mg/mL) in well-degassed PB (0.1 M, pH 7.0) containing 2 mM ethylenediaminetetraacetic acid (EDTA) was allowed to react with a 1000-fold molar excess of 2-mercaptoethanol (2-ME) at room temperature for 30 min. Excess 2-ME was removed by a centrifuged column procedure (29) using Sephadex G-50 (Pharmacia Biotech, Tokyo, Japan) equilibrated and eluted with PB (0.1 M, pH 6.0) containing 2 mM EDTA. Aliquots of this mixture were sampled for estimation of the number of thiol groups with 2,2′-dithiopyridine (30). The filtrate (215 µL) was then added to a reaction vial containing crude [125/131I]MIH. After the reaction mixture was agitated gently for 2 h at room temperature, 39 µL of iodoacetamide (10 mg/mL) in PB (0.1 M, pH 6.0) was added. The reaction mixture was further incubated for 30 min to alkylate the unreacted thiol groups (8). [125/131I]MIH-labeled OST7 (IgG) was subsequently purified by the centrifuged column procedure, equilibrated, and eluted with phosphatebuffered saline (PBS; 0.1 M, pH 6.0). Conjugation of Fab fragment of OST7 with [131I]MIH was performed by reaction of crude [131I]MIH with OST7 (Fab) (410 µL, 1.4 mg/mL) freshly pretreated with a 1000fold molar excess of 2-ME to expose thiol groups. [131I]MIH was prepared according to the procedure described above. After incubation of the reaction mixture for 2 h at room temperature, iodoacetamide (37 µL; 10 mg/mL) was added. [131I]MIH-labeled OST7 (Fab) was purified according to the centrifuged column procedure as described above. Preparation of [125/131I]SIB-Labeled OST7 (IgG and Fab). [125/131I]SIB was prepared by the radioiodination of ATE with Na[125/131I]I according to the procedure described above except that 0.45 mg/mL ATE in 1% acetic acid-methanol was used in place of MIH. Radiochemical yields of [125/131I]SIB were determined by TLC developed with a mixture of 30% ethyl acetate in hexane (6). Conjugation of [125/131I]SIB with OST7s (IgG and Fab) was performed according to the procedure of Zalutsky et al. (6) with slight modifications as follows: A solution of OST7 (IgG; 120 µL, 2.5 mg/mL) or OST7 (Fab; 200 µL, 4 mg/mL) in 0.2 M borate buffer (pH 8.5) was added to the dried residue of crude [125/131I]SIB. After gentle incubation for 1 h at room temperature, radioiodinated OST7s were purified according to the centrifuged column procedure, equilibrated, and eluted with PB (0.1 M, pH 7.4). Preparation of 125I-OST7 (IgG). Direct radioiodination of OST7 (IgG) was performed according to the chloramine T method (31). To 200 µL aliquots of OST7

Arano et al.

(0.25 mg/mL) in 0.3 M PB (pH 7.5) was added 5 µL of Na[125I]I. Chloramine T (0.1 mg/mL, 25 µL), freshly prepared in the same buffer, was then added. After incubation of the mixture for 10 min at room temperature, the reaction was terminated by addition of 6 µL of aqueous sodium bisulfite (0.7 mg/mL). 125I-Labeled OST7 (IgG) was then purified according to the centrifuged column procedure. Radiochemical purities of the radioiodinated mAbs were determined by size-exclusion HPLC, TLC, and cellulose acetate electrophoresis (CAE). CAE was run at an electrostatic field of 0.8 mA/cm for 45 min in the veronal buffer (I ) 0.05, pH 8.6). Preparation of 111In-Labeled OST7 (IgG). OST7 (IgG) was also labeled with 111In using cDTPA or EMCSBz-EDTA as the BCA. cDTPA was conjugated to OST7 according to the procedure of Paik et al. (32) with slight modifications. To a solution of OST7 (400 µL; 6 mg/mL) in 0.05 M borate-buffered saline (pH 8.5) was added 20 µL of cDTPA in dry dimethyl sulfoxide (5.7 mg/mL). After gentle stirring at room temperature for 30 min, a small aliquot (10 µL) was removed for 111In-radiolabeling (see below) to estimate the number of DTPA molecules attached per molecule of OST7. The remainder of the reaction mixture was purified by size-exclusion HPLC, followed by Sephadex G-50 column chromatography (1.8 × 40 cm), equilibrated, and eluted with 20 mM 2-(Nmorpholino)ethanesulfonic acid (MES)-buffered saline (pH 6.0). EMCS-Bz-EDTA was conjugated to OST7 (IgG) by reducing the disulfide bonds in OST7 according to a procedure similar to that used for [131I]MIH conjugation. EMCS-Bz-EDTA-OST7 (IgG) was purified from unreacted small molecular weight compounds according to the centrifuged column procedure using Sephadex G-50, equilibrated, and eluted with 20 mM MES-buffered saline (pH 6.0). The number of EMCS-Bz-EDTA introduced per molecule of OST7 was determined by measuring the thiol groups before and after conjugation reaction using 2,2′dithiopyridine (30), as previously reported (8). The purified cDTPA-OST7 (IgG) and EMCS-Bz-EDTAOST7 (IgG) were labeled with 111In according to the procedure of Brechbiel et al. (4) with slight modifications as previously described (15). Briefly, 5 µL of 111InCl3 in 0.02 N HCl was added to a mixed solution (5 µL) of 1 M sodium acetate and 1.75 N HCl (4:1). After 3 min, 40 µL of respective conjugate (1 mg/mL) was added, and the reaction solution was kept at 23 °C for 1 h. A 13 µL aliquot of 2 mM EDTA in 0.1 M acetate buffer (pH 5.0) was then added to each reaction solution. After 30 min of incubation at 23 °C, 111In-labeled OST7s were purified according to the centrifuged column procedure using Sephadex G-50, equilibrated with 0.1 M PBS (pH 6.0). cDTPA-OST7 (IgG) was further purified by size-exclusion HPLC under the conditions described above. Radiochemical purity of 111In-cDTPA-OST7 (IgG) was determined by CAE and TLC developed with a mixture of 10 % aqueous ammonium formate-methanol-0.2 M aqueous citric acid (2:2:1). The number of cDTPA per OST7 molecule was also determined by the 111In radiolabeling of an unpurified reaction solution of cDTPAOST7 (IgG), followed by TLC analysis, as previously described (33, 34). The radiochemical purity of 111InEMCS-Bz-EDTA-OST7 (IgG) was determined by CAE. Immunoreactivity Measurement. The immunoreactivity of radioiodinated OST7s (IgG) was determined as described previously (8, 25). Tumor cells (2 × 104-1 × 107) suspended in 100 µL of Dulbecco’s PBS were incubated with 100 µL of radiolabeled OST7 in microcentrifugation tubes (5.7 × 46 mm) for 2 h at 4 °C. After centrifugation at 10000g for 5 min, the supernatant was

Bioconjugate Chem., Vol. 7, No. 6, 1996 631

Metabolizable Ester Bond in Radioimmunoconjugates Scheme 1

discarded and the radioactivity determined using a well counter (ARC 2000, Aloka, Tokyo, Japan). Stability Estimation of the Ester Bonds in [131I]MIH-OST7s (IgG and Fab). [131I]MIH-OST7s (IgG and Fab) were diluted to 0.5 mg/mL with 0.1 M PBS (pH 6.0), and 20 µL of each solution was added to 230 µL of freshly prepared murine plasma, human serum, or 20 mM PBS (pH 7.4). After incubation for 1, 3, 6, or 24 h at 37 °C, samples were taken from the solutions, and the percentages of radioactivity released from each antibody were analyzed by paper chromatography (PC; No. 50, Advantec Toyo, Tokyo, Japan) and TLC. Each value was calculated by dividing the antibody-bound radioactivity at various intervals by the radiochemical purity of freshly prepared respective antibody. PC was developed with a mixture of methanol-water (8:2), and TLC was developed with a mixture of chloroform-methanol-water (15: 8:1). Under these analytical conditions, protein-bound radioactivity, free iodine, m-iodobenzoic acid, and miodohippuric acid had Rf values of 0, 0.6-0.65, 0.9-0.95, and 0.9-0.95 on PC and 0, 0.1, 0.4, and 0.25 on TLC, respectively. Percentages of radioactivity released from [125I]SIB-OST7 (IgG and Fab) were also determined under similar conditions. In Vivo Studies. Each radiolabeled OST7 was diluted to 200 µg/mL with 0.1 M PBS (pH 6.0). Two-milliliter aliquots of [125I]MIH-OST7 (IgG) were mixed with the same volume of [131I]SIB-OST7 (IgG) prior to administration. [125I]MIH-OST7 (IgG) was also mixed with the same volume of 111In-cDTPA-OST7 (IgG) or 111In-EMCSBz-EDTA-OST7 (IgG) for in vivo studies. Biodistributions of radioactivity after intravenous administration of each mixture in 6-week-old male ddY mice (35) were monitored at 1, 3, 6, 24, 48, and 72 h postinjection. Groups of five mice, each receiving 20 µg of radiolabeled OST7, were used for the experiments. Organs of interest were removed and weighed, and the radioactivity was determined with a well counter (ARC 2000). At 24 h postinjection of [125I]MIH-OST7 (IgG), urine samples were collected and analyzed by TLC, RP-HPLC, and PC as described above. In RP-HPLC, free iodine, m-iodohippuric acid, and m-iodobenzoic acid had retention times of 2.9, 9.4, and 30.8 min, respectively. Biodistributions of radioactivity after injection of a mixed solution of [131I]MIH-OST7 (Fab) and [125I]SIBOST7 (Fab) (10 µg each) were determined at 10 and 30 min and 1, 3, 6, and 24 h postinjection according to the procedures described above. Athymic mice bearing osteogenic sarcoma were also

treated intravenously with a mixture of [125I]SIB-OST7 (IgG) and [131I]MIH-OST7 (IgG) (10 µg each), and the animals were sacrificed at 24 and 48 h postinjection (five mice each group). Mean weights of mice and tumors used in this study were 20.30 ( 0.48 and 0.44 ( 0.13 g, respectively. Organs of interest were removed and weighed, and the radioactivity in each tissue was determined (ARC-2000). Statistical Analysis. Data are expressed as means ( standard deviation where appropriate. Results were statistically analyzed using unpaired t-test for in vitro studies and paired t-test for in vivo studies. Differences were considered statistically significant when the p value was less than 0.05. RESULTS

Preparation of Radiolabeled OST7s. Radioiodination of MIH was performed in 1% AcOH-MeOH in the presence of NCS as an oxidant (Scheme 1). After 45 min of reaction at room temperature, [125/131I]MIH was obtained with a radiochemical yield of more than 83% as determined by TLC. A similar procedure was employed for the radioiodination of ATE, which produced [125/131I]SIB with a radiochemical yield of over 80%. All of the radioiodinated compounds were treated with sodium bisulfite to quench any residual NCS, preventing the iodine species from attaching to tyrosine residues of the antibody molecule. Neutralization of NCS also prevented exposure of the protein to the oxidant. Conjugation of [125/131I]MIH with OST7s was performed by reactions of thiolated antibodies with the maleimide group of [125/131I]MIH. 2-ME reduction of OST7 (IgG) and Fab exposed 6.4 and 2.3 molecules of the thiol groups per molecule of OST7 (IgG) and OST7 (Fab), respectively, and subsequent conjugation reactions with [125/131I]MIH were performed at a maleimide to thiol molar ratio of 1:1. The radiochemical yields of [131I]MIH-labeled OST7 (IgG) and OST7 (Fab) were 50.2 and 63.7%, respectively, as determined by CAE. After purification by the centrifuged column procedure, [131I]MIH-OST7 (IgG) and [131I]MIH-OST7 (Fab) were obtained with respective radiochemical purities of 92.7 and 93.5% for subsequent studies. [131I]MIH-OST7 (IgG) and [131I]MIH-OST7 (Fab) showed specific activities of 55.5 MBq (1.5 mCi)/ mg and 12.1 MBq (0.33 mCi)/mg, respectively. Sizeexclusion HPLC analyses of [131I]MIH-labeled OST7 (IgG) and OST7 (Fab) indicated more than 96.2 and 95.5% radioactivities in fractions with retention times of 15.4

632 Bioconjugate Chem., Vol. 7, No. 6, 1996

Arano et al. Table 1. Percent Radioactivity in Protein Fractions following Incubation of [131I]MIH-OST7 (IgG) and [131I]MIH-OST7 (Fab) in Human Serum, Murine Plasma, and Buffered Solutiona incubation time (h) 1

[131I]MIH-OST7 (IgG) [131I]MIH-OST7 (Fab) serum plasma buffer serum plasma buffer

97.15 (0.17) 3 90.66 (0.51) 6 79.81 (1.04) 24 43.44 (5.58) half-life (days) 0.87 (0.09)

98.68 (0.47) 96.12 (0.05) 89.10 (0.94) 66.40 (1.13) 1.47 (0.05)

99.80 (0.49) 97.12 (0.73) 95.89 (1.06) 82.50 (3.84) 2.91 (0.57)

94.33 (1.56) 81.68 (0.41) 66.80 (2.76) 27.22 (3.44) 0.62 (0.02)

97.69 (0.30) 91.92 (1.50) 81.43 (2.11) 54.46 (1.08) 1.08 (0.03)

99.26 (0.29) 97.01 (1.42) 96.47 (1.15) 83.78 (1.02) 2.94 (0.29)

a [131I]MIH-OST7 (IgG) or [131I]MIH-OST7 (Fab) (20 µL each) was added to 230 µL of human serum, murine plasma, or buffered solution (20 mM PBS, pH 7.4) at 37 °C. Expressed as means (SD) from four experiments.

Figure 2. Size-exclusion HPLC profiles of the radioactivity of [131I]MIH-OST7 (IgG) (A) and [131I]MIH-OST7 (Fab) (B). More than 96% (A) and 95% (B) of the radioactivities were observed in fractions similar to those of unmodified OST7 (IgG) and OST7 (Fab), respectively. Under these conditions, thyrogloglin (670 000), OST7 (IgG), bovine serum albumin (68 000), OST7 (Fab), and cytochrome c (13 000) were eluted at retention times of 11.4, 15.4, 16.8, 18.8, and 21.0 min, respectively.

and 18.8 min, respectively, which were similar to those of the original (nontreated) OST7 (IgG) and OST7 (Fab), respectively (Figure 2). Under similar conditions, [125I]MIH-OST7 (IgG) was also prepared with radiochemical purity and specific activity of 98.1% and 10.36 MBq (0.28 mCi)/mg, respectively. Conjugation of [131I]SIB with OST7 (IgG) and OST7 (Fab) was performed at active ester to protein molar ratios of 7:1 and 4:1, respectively, with radiochemical yields of 47.5 and 55.5%. The radiochemical purities and specific activities of the resulting [131I]SIB-labeled mAbs were 99.3% and 52 MBq (1.4 mCi)/mg for OST7 (IgG) and 93.5% and 11.8 MBq (0.32 mCi)/mg for OST7 (Fab), respectively. [125I]SIB-OST7 (IgG) was also prepared according to similar procedures with a radiochemical purity and a specific activity of 97.0% and 12.2 MBq (0.33 mCi)/mg, respectively. Direct radioiodination of OST7 (IgG) generated 125I-labeled OST7 (IgG) with a radiochemical purity and a specific activity of 97.3% and 92.5 MBq (2.5 mCi)/mg, respectively. OST7-cDTPA was prepared by reaction of acid anhydride with amine residues of the protein. 111In radiolabeling of unpurified reaction solution and subsequent analysis by TLC estimated the number of cDTPA per molecule of OST7 to be 2.2. The radiochemical purity of 111 In-cDTPA-OST7 was 99.4 and 68.6% when analyzed by TLC and CAE, respectively. Since further attempts to improve the radiochemical purity were unsuccessful (36), 111In-cDTPA-OST7 (IgG) of this purity was used for subsequent in vivo study. EMCS-Bz-EDTA-OST7 (IgG) conjugate was prepared by maleimide-thiol chemistry using the maleimide group of EMCS-Bz-EDTA and the thiol groups of OST7 (IgG), generated by reducing

the disulfide bonds. The number of EMCS-Bz-EDTA molecules attached per molecule of OST7 was 2.2 by measuring the thiol groups before and after conjugation reaction. The radiochemical purity of 111In-EMCS-BzEDTA-OST7 (IgG) after the centrifuged column procedure was 95.9% as determined by CAE. Stability of the Ester Bond in [131I]MIH-OST7s (IgG and Fab). Stabilities of the ester bonds in both [131I]MIH-OST7 (IgG) and [131I]MIH-OST7 (Fab) were estimated in murine plasma, human serum, and buffered solution of neutral pH (pH 7.4) at 37 °C. Table 1 shows the percentages of the protein-bound radioactivity of each [131I]MIH-OST7 at 1, 3, 6, and 24 h postincubation as determined by TLC analysis. Both [131I]MIH-OST7 (IgG) and [131I]MIH-OST7 (Fab) released significantly more radioactivity in human serum than in murine plasma, with the lowest level of release in the buffered solution. In addition, while no significant differences were observed in the radioactivity release from the two [131I]MIH-labeled OST7s in the buffered solution, [131I]MIH-OST7 (Fab) liberated significantly more radioactivity than [131I]MIH-OST7 (IgG) in both human serum and murine plasma. The calculated half-lives of [131I]MIH-OST7 (IgG) and [131I]MIH-OST7 (Fab) in the respective media are also presented in Table 1. The radioactivity released from the two [131I]MIH-labeled OST7s showed Rf values on TLC and PC analyses similar to those of m-iodohippuric acid. Under similar experimental conditions, both [125I]SIB-OST7 (IgG) and [125I]SIB-OST7 (Fab) released 96.6%) was detected at a retention time (A) or a Rf value (B) identical to those of m-iodohippuric acid by both analyses.

results indicated that the ester bond in [125I]MIH-OST7 (IgG) had higher stability against esterase-mediated hydrolysis in vivo than that observed in vitro. This was also supported by the similar radioactivity levels in the tumor at 24 h postinjection of [131I]MIH-OST7 (IgG) and [125I]SIB-OST7 (IgG) in athymic mice (Table 2). The differences in stability of the ester bond in [125I]MIHOST7 (IgG) in vitro and in vivo may be attributable to the rapid blood stream in vivo, which may have hindered esterase access to its substrate. These results also suggested that the rapid hydrolysis of the ester bond in

[125I]MIH-OST7 (IgG) in human serum would not predict rapid cleavage of the ester bond when administered to humans. [125I]MIH-OST7(IgG) showed rapid systemic clearance of the radioactivity with over 96% of the excreted radioactivity being recovered in the urine. Analysis of urine confirmed selective release of m-iodohippuric acid (Figure 5). Low radioactivity levels in the stomach along with negligible amounts of free radioiodine in the urine following administration of [125I]MIH-OST7 (IgG) also indicated that in vivo deiodination was not involved in the rapid systemic clearance of radioactivity by the radioimmunoconjugate. Since radiolabeled mAbs are rapidly metabolized into smaller molecular weight radiometabolites in nontarget tissues (33, 40, 41), lower radioactivity levels in nontarget tissues with [125I]MIHOST7 (IgG) would be attributable to faster elimination

Bioconjugate Chem., Vol. 7, No. 6, 1996 635

Metabolizable Ester Bond in Radioimmunoconjugates

Figure 7. Tumor-to-organ ratios of the radioactivity at 24 and 48 h postinjection of [131I]MIH-OST7 (IgG) (solid column) and [125I]SIB-OST7 (IgG) (hatched column) in athymic mice bearing osteogenic sarcoma. Table 2. Biodistribution of Radioactivity after Simultaneous Administration of [131I]MIH-OST7 (IgG) and [125I]SIB-OST7 (IgG) in Athymic Mice Bearing Osteogenic Sarcomaa,b [131I]MIH-OST7 (IgG) tissue

24 h

48 h

[125I]SIB-OST7 (IgG) 24 h

48 h

blood 8.74 (0.79) 4.66 (0.52) 12.51 (1.56) 8.98 (1.30) liver 2.48 (0.14) 0.82 (0.16) 3.72 (0.32) 1.72 (0.33) kidneys 2.43 (0.84) 1.23 (0.07) 3.70 (0.32) 2.78 (0.09) intestine 0.68 (0.07) 0.32 (0.03) 1.09 (0.10) 0.68 (0.04) spleen 1.69 (0.09) 0.79 (0.16) 2.32 (0.32) 1.87 (0.59) lungs 3.58 (0.35) 1.78 (0.14) 5.19 (0.80) 3.46 (0.36) stomachc 0.23 (0.06) 0.12 (0.05) 0.27 (0.06) 0.21 (0.05) neckc 0.18 (0.04) 0.17 (0.03) 0.22 (0.08) 0.31 (0.07) tumor 22.89 (3.39)d 16.60 (3.94) 23.47 (4.42)d 21.30 (5.42) a Mean weights of mice and tumors were 20.30 ( 0.48 g and 0.44 ( 0.13 g, respectively. b Expressed as percent of injected dose per gram of wet tissue. c Expressed as percent of injected dose per tissue. d Not significantly different by paired t-test.

rates of the radiometabolite derived from [125I]MIHOST7 (IgG), m-iodohippuric acid, compared with those from [131I]SIB-OST7 (IgG) and 111In-EMCS-Bz-EDTAOST7 (IgG), although there would also be some contribution of faster blood clearance of [125I]MIH-OST7 (IgG). This was more clearly demonstrated by the different radioactivity levels in the tissues such as the liver and kidney between [125I]MIH-OST7 (IgG) and 111In-cDTPAOST7 (IgG). While [125I]MIH-OST7 (IgG) demonstrated higher radioactivity levels in the blood than 111In-cDTPAOST7 (IgG) throughout the experimental period, the latter demonstrated significantly higher radioactivity levels in the liver and kidney after 24 h postinjection. Although nonchelated 111In-labeled species in 111IncDTPA-OST7 (IgG) would be partially responsible for high radioactivity levels in these tissues, higher and persistent radioactivity levels in the liver, kidney, and spleen along with the lower radioactivity levels in the blood with 111In-cDTPA-OST7 (IgG) than with [125I]MIH-OST7 (IgG) reinforced the important role played by the radiometabolites in the elimination of radioactivity from nontarget tissues, as documented previously (15, 17, 18, 23). Thus, these findings indicated that radiochemical design of mAbs that liberates radiometabolites

with rapid urinary excretion could improve the undesirable localization of radioactivity in nontarget tissues. In biodistribution studies in athymic mice, [131I]MIHOST7 (IgG) rendered radioactivity levels in the tumor and nontarget such as the liver and kidney similar to and significantly lower than those of simultaneously administered [125I]SIB-OST7 (IgG), respectively (Table 2). These results also supported the preferential release of m-iodohippuric acid from [125I]MIH-OST7 (IgG) in nontarget tissues. However, although [131I]MIH-OST7 (IgG) indicated target-to-nontarget ratios of radioactivity significantly higher than those of [125I]SIB-OST7 (IgG) at both 24 and 48 h postinjection (Figure 7), the former showed a small but significant decrease in the target radioactivity level at 48 h postinjection when compared with the latter (Table 2). No significant decrease in the radioactivity level in the tumor was observed with [125I]SIB-OST7 (IgG) between the postinjection intervals. In our previous study using NGA, although [131I]SIB-NGA was eliminated from murine liver more slowly than [131I]MIH-NGA, the former registered residual radioactivity levels in the liver similar to the latter at 24 h postinjection (19). In addition, radiolabeled OST7 (IgG) via direct radioiodination (26, 42), via [67Ga]succinyldeferoxamine with an ester bond (8), and via [111In]-cDTPA (43) indicated unchanged radioactivity levels in the same tumor model (KT005) during the same postinjection intervals. Therefore, it is likely that the decreased radioactivity level in the tumor at 48 h postinjection of [131I]MIH-OST7 (IgG) was caused by gradual cleavage of the ester bond even when the radioimmunoconjugate was bound to the antigen on the target cell surface, although cleavage of the ester bond in the blood may have also been involved. The slower elimination rate of radioactivity from the tumor than from blood, as reflected in the increased target-to-blood ratio with time (Figure 7), would be attributable to the formation of antigenantibody complexes at the tumor site, which might have exerted additional steric interference against esterase access to its substrate. However, such steric interference on tumor cells would have little effect on chemical hydrolysis of the ester bond. This was supported by the

636 Bioconjugate Chem., Vol. 7, No. 6, 1996

similar hydrolysis rates of [131I]MIH-conjugated OST7 (IgG) and OST7 (Fab) in the buffered solution (Table 1). These findings suggested that while MIH may be applicable for diagnostic purposes in combination with 123I, this reagent may not be suitable for therapeutic applications. When MIH was applied to Fab fragment, the ester bond in [131I]MIH-OST7 (Fab) was found to be much more labile against esterase-mediated hydrolysis than that in [131I]MIH-OST7 (IgG) (Table 1). Such labile characteristics of the ester bond in [131I]MIH-OST7 (Fab) would be primarily responsible for the rapid systemic clearance of its radioactivity (Figure 6), although partial dissociation of noncovalently bonded chains in blood would also be involved. These results suggested that the steric interference induced by the protein molecule plays a critical role in the stability of the ester bond in MIHconjugated polypeptides and that MIH would be less applicable to conjugation of Fab or smaller molecular weight polypeptides. In conclusion, the present study indicated that use of a metabolizable linkage between mAbs and radiolabeled compound with rapid urinary excretion characteristics constitutes an attractive strategy to decrease the radioactivity levels in nontarget tissues. Application of 123Ilabeled-MIH to IgG mAb is promising for tumor diagnosis at early postinjection times. However, this study also indicated problems associated with the use of an ester bond as the metabolizable linkage due to its labile characteristics and limited applicability to larger molecular weight proteins. These results call for development of a new class of metabolizable linkage that allows stable attachment of the radiolabel in plasma but liberates designed radiometabolites with carboxylate from covalently conjugated proteins following lysosomal proteolysis in nontarget tissues. Combination of such linkages with radiolabeled compounds with rapid urinary excretion characteristics would further enhance targetto-nontarget ratios of the radioactivity with various radiopharmaceuticals derived from not only mAbs of high molecular weight but also peptides of rather lower molecular weight. ACKNOWLEDGMENT

We thank Dr. Isamu Yomoda, Daiichi Radioisotope Laboratories Ltd., Tokyo, Japan, for providing Na[131I]I. We are grateful to Nihon Medi-Physics for their kind gift of 111InCl3. This work was supported in part by Grantsin-Aid for Developing Scientific Research (07672419 for Y.A. and 07274108 for A.Y.) from the Ministry of Education, Science and Culture of Japan. LITERATURE CITED (1) Co, M. S., Yano, S., Hsu, R. K., Landolfi, N. F., Vasquez, M., Cole, M., Tso, J. T., Bringman, T., Laird, W., Hudson, D., Kawamura, K., Suzuki, K., Furuichi, K., Queen, C., and Masuho, Y. (1994) A Humanized Antibody Specific for the Platelet Integrin GpIIb/IIIa. J. Immunol. 152, 2968-2976. (2) 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 CA-125 for Radioimmunoimaging of Ovarian Cancer. Cancer Immunol. Immunother. 37, 143-149. (3) Yokota, T., Milenic, D. E., Whitlow, M., and Schlom, J. (1992) Rapid Tumor Penetration of a Single-Chain Fv and Comparison with Other Immunoglobulin Forms. Cancer Res. 52, 3402-3408. (4) Brechbiel, M. W., Gansow, O. A., Atcher, R. W., Schlom, J., Esteban, J., Simpson, D. E., and Colcher, D. (1986) Synthesis of 1-(p-isothiocyanatobenzyl) Derivatives of DTPA and EDTA.

Arano et al. Antibody Labeling and Tumor-Imaging Study. Inorg. Chem. 25, 2772-2781. (5) Subramanian, R., Colony, J., Shaban, S., Sidrak, H., Haspel, M. V., Pomato, N., Hanna, M. G., and Mccabe, R. P. (1992) New Chelating Agent for Attaching Indium-111 to Monoclonal AntibodiessInvitro and Invivo Evaluation. Bioconjugate Chem. 3, 248-255. (6) Zalutsky, M. R., and Narula, A. S. (1987) A Method for the Radiohalogenation of Proteins Resulting in Decreased Thyroid Uptake of Radioiodine. Appl. Radiat. Isot. 38, 1051-1055. (7) Pietersz, G. A., and Krauer, K. (1994) Antibody-Targeted Drugs for the Therapy of Cancer. J. Drug Targeting 2, 183215. (8) Arano, Y., Inoue, T., Mukai, T., Wakisaka, K., Sakahara, H., Konishi, J., and Yokoyama, A. (1994) Discriminated Release of a Hippurate-Like Radiometal Chelate in Nontarget Tissues for Target-Selective Radioactivity Localization Using pH-Dependent Dissociation of Reduced Antibody. J. Nucl. Med. 35, 326-333. (9) Faivre-Chauvet, A., Gestin, J. F., Mease, R. C., Sai-Maurel, C., Thedrez, P., Slinkin, M., Meinken, G. E., Srivastava, S. C., and Chatal, J. F. (1993) Introduction of five Potentially Metabolizable Linking Groups Between In-111-Cyclohexyl EDTA Derivatives and F(ab′)2 Fragments of Anti-Carcinoembryonic Antigen Antibody. 2. Comparative Pharmacokinetics and Biodistribution in Human Colorectal CarcinomaBearing Nude Mice. Nucl. Med. Biol. 20, 763-771. (10) Haseman, M. K., Goodwin, D. A., Meares, C. F., Kaminski, M. S., Wensel, T. G., McCall, M. J., and Levy, R. (1986) Metabolizable 111In chelate conjugated anti-idiotype monoclonal antibody for radioimmunodetection of lymphoma in mice. Eur. J. Nucl. Med. 12, 455-460. (11) Meares, C. F., McCall, M. J., Deshpande, S. V., DeNardo, S., and Goodwin, D. A. (1988) Chelate Radiochemistry: Cleavable Linkers Lead to Altered Levels of Radioactivity in the Liver. Int. J. Cancer, Suppl. 2, 99-102. (12) Paik, C. H., Yokoyama, K., Reymonds, J. C., Quadri, S. M., Min, C. Y., Shin, S. Y., Maloney, P. J., Larson, S. M., and Reba, R. C. (1989) Reduction of Background Activities by Introduction of a Diester Linkage Between Antibody and a Chelate in Radioimmunodetection of Tumor. J. Nucl. Med. 30, 1693-1701. (13) Quadri, S. M., Vriesendorp, H. M., Leichner, P. K., and Williams, J. R. (1993) Evaluation of Indium-111-Labeled and Yttrium-90-Labeled Linker-Immunoconjugates in Nude Mice and Dogs. J. Nucl. Med. 34, 938-945. (14) Weber, R. W., Boutin, R. H., Nedelman, M. A., ListerJames, J., and Dean, R. T. (1990) Enhanced Kidney Clearance with an Ester-Linked 99mTc-Radiolabeled Antibody Fab′Chelator Conjugate. Bioconjugate Chem. 1, 431-437. (15) Arano, Y., Mukai, T., Uezono, T., Wakisaka, K., Motonari, H., Akizawa, H., Taoka, Y., and Yokoyama, A. (1994) A biological method to evaluate bifunctional chelating agents to label antibodies with metallic radionuclides. J. Nucl. Med. 35, 890-898. (16) Arano, Y., Mukai, T., Akizawa, H., Uezono, T., Motonari, H., Wakisaka, K., Kairiyama, C., and Yokoyama, A. (1995) Radiolabeled Metabolites of Proteins Play a Critical Role in Radioactivity Elimination from the Liver. Nucl. Med. Biol. 22, 555-564. (17) Duncan, J. R., and Welch, M. J. (1993) Intracellular Metabolism of Indium-111-DTPA-Labeled Receptor Targeted Proteins. J. Nucl. Med. 34, 1728-1738. (18) Franano, F. N., Edwards, W. B., Welch, M. J., and Duncan, J. R. (1994) Metabolism of Receptor Targeted 111In-DTPAGlycoproteins: Identification of 111In-DTPA--lysine as the Primary Metabolic and Excretory Product. Nucl. Med. Biol. 21, 1023-1034. (19) Arano, Y., Wakisaka, K., Ohmomo, Y., Uezono, T., Mukai, T., Motonari, H., Shiono, H., Sakahara, H., Konishi, J., Tanaka, C., and Yokoyama, A. (1994) Maleimidoethyl 3-(trin-butylstannyl)hippurate: A useful radioiodination reagent for protein radiopharmaceuticals to enhance target selective radioactivity localization. J. Med. Chem. 37, 2609-2618. (20) Boyle, C. C., Paine, A. J., and Mather, S. J. (1992) The Mechanism of Hepatic Uptake of a Radiolabelled Monoclonal Antibody. Int. J. Cancer 50, 912-917.

Bioconjugate Chem., Vol. 7, No. 6, 1996 637

Metabolizable Ester Bond in Radioimmunoconjugates (21) Sands, H., and Jones, P. L. (1987) Methods for the Study of the Metabolism of Radiolabeled monoclonal Antibodies by Liver and Tumor. J. Nucl. Med. 28, 390-398. (22) Deshpande, S. V., Subramanian, R., McCall, M. J., DeNardo, S. J., DeNardo, G. L., and Meares, C. F. (1990) Metabolism of Indium Chelates Attached to Monoclonal Antibody: Minimal Transchelation of Indium from BenzylEDTA chelate In Vivo. J. Nucl. Med. 31, 218-224. (23) Arano, Y., Mukai, T., Uezono, T., Motonari, H., Wakisaka, K., and Yokoyama, A. (1994) Biological Comparison of DTPA and SCN-Bz-EDTA as a Chelating Agent for Indium Labeling of Antibodies. J. Labelled Compd. Radiopharm. 35, 381-383. (24) Garg, P. K., Alston, K. L., and Zalutsky, M. R. (1995) Catabolism of radioiodinated murine monoclonal antibody F(ab′)2 fragment labeled using N-succinimidyl 3-iodobenzoate and iodogen methods. Bioconjugate Chem. 6, 493-501. (25) Koizumi, M., Endo, K., Kunimatsu, M., Sakahara, H., Nakashima, T., Kawamura, Y., Watanabe, Y., Saga, T., Konishi, J., Yamamuro, T., Hosoi, S., Toyama, S., Arano, Y., and Yokoyama, A. (1988) 67Ga-labeled Antibodies for Immunoscintigraphy and Evaluation of Tumor Targeting of DrugAntibody Conjugate in Mice. Cancer Res. 48, 1189-1194. (26) Sakahara, H., Endo, K., Koizumi, M., Nakashima, T., Kunimatsu, M., Watanabe, Y., Kawamura, Y., Nakamura, T., Ohta, H., Kotoura, Y., Yamamuro, T., Hosoi, S., Toyama, S., and Torizuka, K. (1988) Relationship Between In Vitro Binding Activity and In Vivo Tumor Accumulation of Radiolabeled Monoclonal Antibodies. J. Nucl. Med. 29, 235-240. (27) Hosoi, S., Nakamura, T., Higashi, S., Yamamuro, T., Toyama, S., Shinimiya, K., and Mikawa, H. (1982) Detection of human osteosarcoma-associated antigen(s) by monoclonal antibodies. Cancer Res. 42, 654-659. (28) Rousseaux, J., Rousseaux-Prevost, R., and Bazin, H. (1986) Optimal Conditions for the Preparation of Proteolytic Fragments from Monoclonal IgG of Different Rat IgG Subclasses. Methods Enzymol. 121, 663-669. (29) Meares, C. F., McCall, M. J., Reardan, D. T., Goodwin, D. A., Diamanti, C. I., and McTigue, M. (1984) Conjugation of Antibodies with Bifunctional Chelating Agents: Isothiocyanate and Bromoacetamide Reagents, Methods of Analysis, and Subsequent Addition of Metal Ions. Anal. Biochem. 142, 68-78. (30) Grassetti, D. R., and Murray, Jr., J. F. (1967) Determination of Sulfhydryl Groups with 2,2′- or 4,4′-Dithiodipyridine. Arch. Biochem. Biophys. 119, 41-49. (31) Wilbur, D. S., Hadley, S. W., Grant, L. M., and Hylarides, M. D. (1991) Radioiodinated Iodobenzoyl Conjugates of a Monoclonal Antibody Fab Fragment. In Vivo Comparisons with Chloramine-T-Labeled Fab. Bioconjugate Chem. 2, 111116. (32) Paik, C. H., Hong, J. J., Ebbert, M. A., Heald, S. C., Reba, R. C., and Eckelman, W. C. (1985) Relative Reactivity of DTPA, Immunoreactive Antibody-DTPA Conjugates, and Nonimmunoreactive Antibody-DTPA Conjugates Toward Indium-111. J. Nucl. Med. 26, 482-487. (33) Paik, C. H., Sood, V. K., Le, N., Cioloca, L., Carrasquillo, J. A., Reynolds, J. C., Neumann, R. D., and Reba, R. C. (1992)

Radiolabeled Products in Rat Liver and Serum After Administration of Antibody-Amide-DTPA-Indium-111. Nucl. Med. Biol. 19, 517-522. (34) Sakahara, H., Endo, K., Nakashima, T., Koizumi, M., Ohta, H., Furukawa, T., Ohmomo, Y., Yokoyama, A., Okada, K., Yoshida, O., and Nishi, S. (1985) Effect of DTPA Conjugation on the Antigen Binding Activity and Biodistribution of Monoclonal Antibodies Against alpha-Fetoprotein. J. Nucl. Med. 26, 750-755. (35) Imai, S., Morimoto, J., Tsubura, T., Esaki, K., Michalides, R., Holmes, R. S., Deimling, O., and Hilgers, J. (1986) Genetic marker patterns and endogenous mammary tumor virus genes in inbred mouse strains in Japan. Exp. Anim. 35, 263273. (36) Arano, Y., Uezono, T., Akizawa, H., Ono, M., Wakisaka, K., Nakayama, M., Sakahara, H., Konishi, J., and Yokoyama, A. (1996) Reassessment of Diethylenetriaminepentaacetic Acid (DTPA) as a Chelating Agent for Indium-111 Labeling of Polypeptides Using a Newly Synthesized Monoreactive DTPA Derivative. J. Med. Chem. 39, 3451-3460. (37) Dykes, P. W., Bradwell, A. R., Chapman, C. E., and Vaughan, A. T. N. (1987) Radioimmunotherapy of cancer: clinical studies and limiting factors. Cancer Treat. Rev. 14, 87-106. (38) Gallinger, S., Reilly, R. M., Kirsh, J. C., Odze, R. D., Schmocker, B. J., Hay, K., Polihronis, J., Damani, M. T., Shpitz, B., and Stern, H. S. (1993) Comparative Dual Label Study of First and Second Generation Antitumor-associated Glycoprotein-72 Monoclonal Antibodies in Colorectal Cancer Patients. Cancer Res. 53, 271-278. (39) Bormans, G., Nerom, C. V., Hoogmartens, M., Roo, M. D., and Verbruggen, A. (1989) Metabolism of Tc-99m ECD in Isolated Rat Organs. J. Nucl. Med. 30, 743 (Abstract). (40) Himmelsbach, M., and Wahl, R. L. (1989) Studies on the Metabolic Fate of 111In-labeled Antibodies. Nucl. Med. Biol. 16, 839-845. (41) Motta-Hennessy, C., Sharkey, R. M., and Goldenberg, D. M. (1990) Metabolism of Indium-111-Labeled Murine Monoclonal Antibody in Tumor and Normal Tissue of the Athymic Mouse. J. Nucl. Med. 31, 1510-1519. (42) Kobayashi, H., Sakahara, H., Hosono, M., Yao, Z. S., Toyama, S., Endo, K., and Konishi, J. (1994) Improved clearance of radiolabeled biotinylated monoclonal antibody following the infusion of avidin as a “chase” without decreased accumulation in the target tumor. J. Nucl. Med. 35, 16771684. (43) Sakahara, H., Endo, K., Kunimatsu, M., Kawamura, Y., Ohta, H., Nakamura, T., Tanaka, H., Kotoura, Y., Yamamuro, T., Hosoi, S., Toyama, S., and Torizuka, K. (1987) Localization of Human Osteogenic Sarcoma Xenografts in Nude Mice by a Monoclonal Antibody Labeled with Radioiodine and Indium111. J. Nucl. Med. 28, 342-348.

BC960058W