Assessment of the Radiochemical Design of Antibodies with a

Department of Patho-Functional Bioanalysis, Graduate School of Pharmaceutical Sciences, and Department of. Nuclear Medicine and Diagnostic Imaging, ...
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Bioconjugate Chem. 1998, 9, 497−506

497

Assessment of the Radiochemical Design of Antibodies with a Metabolizable Linkage for Target-Selective Radioactivity Delivery Yasushi Arano,*,† Kouji Wakisaka,† Hiromichi Akizawa,† Masahiro Ono,† Keiichi Kawai,‡ Morio Nakayama,§ Harumi Sakahara,| Junji Konishi,| and Hideo Saji† Department of Patho-Functional Bioanalysis, Graduate School of Pharmaceutical Sciences, and Department of Nuclear Medicine and Diagnostic Imaging, Graduate School of Medicine, Kyoto University, Yoshida-Shimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Central Research Laboratories, Miyazaki Medical College, 5200 Kihara, Kiyotake-cho, Miyazaki-gun 889-1601, and Faculty of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-Honmachi, Kumamoto 862-0973, Japan. Received December 26, 1997; Revised Manuscript Received April 15, 1998

Interposition of a metabolizable linkage has been performed to reduce the hepatic radioactivity levels of radiolabeled antibodies. To estimate the validity of this strategy, a radioiodination reagent (HML) that provides a stable attachment for m-iodohippuric acid with proteins in plasma while facilitating rapid and selective release of the compound after lysosomal proteolysis in the liver was conjugated with a monoclonal antibody (mAb) against osteogenic sarcoma (OST7, IgG1). Radiolabeled OST7 conjugates with a plasma-labile ester bond for releasing m-iodohippuric acid (MIH), plasma-stable amide bonds for releasing radiometabolites of hepatobiliary excretion (MPH), or slow elimination rates from hepatocytes ([111In]EMCS-Bz-EDTA) were prepared with similar conjugation chemistry. The four radiolabeled OST7 conjugates were characterized both in vitro and in vivo. All the radiolabeled OST7 conjugates had similar radiochromatograms on size-exclusion HPLC and similar antigen binding affinities. While MIH-OST7 indicated accelerated clearance of radioactivity from the blood due to the release of m-iodohippurate, the rest of the three radiolabeled OST7 conjugates remained stable in serum incubation studies and had similar radioactivity elimination from the blood in vivo. When injected into normal mice, HML-OST7 demonstrated tissue-to-blood ratios of radioactivity similar to those of MIH-OST7 and significantly lower than those of the other two radiolabeled OST7 conjugates. In biodistribution studies in nude mice, both HML-OST7 and MIH-OST7 exhibited tumor-to-liver or tumor-to-intestine ratios of radioactivity higher than those of [111In]EMCS-Bz-EDTAOST7 or MPH-OST7, respectively. HML-OST7, MPH-OST7, and [111In]EMCS-Bz-EDTA-OST7 indicated there were no changes in the radioactivity levels in the tumor between 24 and 48 h postinjection, whereas MIH-OST7 significantly decreased the radioactivity levels in the tumor at these time points. HML reduced the radioactivity levels in nontarget tissues without impairing the tumor radioactivity levels delivered by OST7. These findings indicated that the design of a radiolabeled mAb that is stable in plasma and liberates the radiometabolite of rapid urinary excretion constitutes an effective strategy for achieving target-selective radioactivity delivery.

INTRODUCTION

The localization of radioactivity in nontarget tissues such as the liver after administration of radiolabeled monoclonal antibodies (mAbs)1 constitutes a serious problem for clinical applications of radiopharmaceuticals. Although the precise mechanisms of mAb accumulation in the liver are still uncertain, target-selective radioactivity localization could be amplified if the radiolabeled mAbs bound to the target remain stable while the radioactivity in nontarget tissues is excreted in the urine. * Address correspondence to Yasushi Arano, Department of Patho-Functional Bioanalysis, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan. Telephone: 81-75-753-4608. Fax: 81-75-753-4568. Email: [email protected]. † Graduate School of Pharmaceutical Sciences, Kyoto University. ‡ Miyazaki Medical College. § Kumamoto University. | Graduate School of Medicine, Kyoto University.

This approach was first reported by Haseman et al., who introduced a diester bond between an antibody and an 111In chelate of benzyl-EDTA as a “metabolizable linkage” to liberate the radiolabeled compound with a low molecular weight after lysosomal proteolysis in nontarget tissues (1). A variety of radiolabeled mAbs with metabolizable linkages have been developed (2-8), and some 1 Abbreviations: mAb, monoclonal antibody; OST7, mAb against osteogenic sarcoma; NGA, galactosylneoglycoalbumin; PB, phosphate buffer; PBS, phosphate-buffered saline; MES, 2-(N-morpholino)ethanesulfonic acid; 2-ME, mercaptoethanol; NCS, N-chlorosuccinimide; HML, 3′-iodohippuryl N-maleoylL-lysine; MIH, maleimidoethyl 3′-iodohippurate; MPH, N-(5maleimidopentyl) 3′-iodohippuric acid amide; SHML, 3′-(tri-nbutylstannyl)hippuryl-N-maleoyl-L-lysine; SMIH, maleimidoethyl 3-(tri-n-butylstannyl)hippurate; SMPH, N-(5-maleimidopentyl) 3′-(tri-n-butylstannyl)hippuric acid amide; EMCS-Bz-EDTA, 1-[4-[(5-maleimidopentyl)amino]benzyl]ethylenediaminetetraacetic acid; HPLC, high-performance liquid chromatography; RP-HPLC, reversed-phase high-performance liquid chromatography; TLC, thin-layer chromatography; CAE, cellulose acetate electrophoresis; PC, paper chromatography.

S1043-1802(97)00220-6 CCC: $15.00 © 1998 American Chemical Society Published on Web 06/19/1998

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studies demonstrated enhanced target-to-nontarget ratios of the radioactivity in animal models. In most studies, ester bonds have been used as the metabolizable linkage of choice due to rapid cleavage of the linkage in nontarget tissues such as the liver and kidney where high esterase activity levels have been reported (9). However, since ester bonds are cleaved by both chemical and enzymatic hydrolyses not only in nontarget tissues but also in plasma, use of ester bonds as the metabolizable linkage impaired the radioactivity levels in the target (1, 5, 10, 11). In addition, while the cleavage of the ester bond in plasma may cause only a slight decrease in the radioactivity levels in the target tissues in animal models, due to the presence of large tumor masses in a small body, the plasma-labile characteristics of ester bonds may significantly decrease the target radioactivity levels in humans where much smaller tumor masses are located in much higher distribution volumes. On the other hand, recent metabolic studies of radiolabeled polypeptides demonstrated that the radiometabolites generated after lysosomal proteolysis of polypeptides play a critical role in the radioactivity levels in the liver (12-17). These findings support the rationale of the radiochemical design of antibodies using a metabolizable linkage to reduce the nontarget radioactivity levels. Furthermore, recent advances in genetic engineering have provided mAbs with less immunogenicity (e.g., humanized mAbs) and more favorable pharmacokinetic characteristics (e.g., single-chain Fv fragment) (18-21). Monoclonal antibodies directed against endothelial cells in solid tumors may also facilitate higher accumulation in target tissues within shorter postinjection intervals (22-24). These classes of mAbs would potentially solve various problems associated with the clinical applications of radiolabeled murine mAbs. These findings and developments prompted us to further assess the validity of the radiochemical design of mAbs with metabolizable linkages for reducing the nontarget radioactivity levels of radiolabeled mAbs. To estimate the validity of the design, the metabolizable linkages should be stable enough in plasma to deliver radioactivity to both target and nontarget tissues equivalent to nonmetabolizable linkages. Furthermore, the radiometabolite generated after cleavage of the metabolizable linkage should be excreted rapidly from the nontarget tissues into urine without redistribution into other tissues. We have recently developed a radioiodination reagent for proteins with L-lysine as the metabolizable linkage, 3′-iodohippuryl N-maleoyl-Llysine (HML). In our previous studies using galactosylneoglycoalbumin (NGA) as a model polypeptide, [131I]HML-NGA selectively liberated m-iodohippuric acid after lysosomal proteolysis in hepatocytes. In addition, the peptide bond of [131I]HML-NGA remained stable in human serum and murine plasma (25). Such chemical and biological characteristics would render HML useful for estimating the validity of the chemical design of mAbs for reducing the nontarget radioactivity localization. In this study, HML was radioiodinated with Na125I or Na131I and conjugated with a mAb against osteogenic sarcoma (OST7, IgG1). For comparison, radioiodinated OST7 conjugates with an ester bond or an amide bond were prepared using maleimidoethyl 3′-iodohippurate (MIH) or N-(5-maleimidopentyl) 3′-iodohippuric acid amide (MPH), respectively. 111In-labeled OST7 was also prepared using 1-[4-[(5-maleimidopentyl)amino]benzyl]ethylenediaminetetraacetic acid (EMCS-Bz-EDTA) as a bifunctional chelating agent. The chemical structures of

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Figure 1. Chemical structures of radiolabeled OST7 conjugates. All the reagents were conjugated with OST7 molecules with maleimide-thiol chemistry after reducing the disulfide bonds in OST7.

radiolabeled OST7 conjugates used in this study are illustrated in Figure 1. All the reagents were attached to OST7 with similar conjugation chemistry using maleimide-thiol chemistry. Prior studies showed that all the reagents provided radiolabeled polypeptides stable against in vivo deiodination or transchelation (14, 2527). Radiometabolites generated after lysosomal proteolysis of radiolabeled NGAs using the respective reagent were well characterized in previous studies (11, 14, 25, 26). While MIH liberates m-iodohippuric acid as the sole radiometabolite in hepatic parenchymal cells at a rate similar to that of HML, the ester bond in MIH is hydrolyzed with time in buffered solutions at neutral pH and in plasma. MPH generates radiometabolites with hepatobiliary excretion after lysosomal proteolysis in the murine liver. NGA-[111In]EMCS-Bz-EDTA generates a cysteine adduct as the predominant radiometabolite in hepatocytes, and the metabolite was eliminated from the liver at a rate that was the slowest of the four reagents. All the radiolabeled OST7 conjugates were characterized in vitro, and the radioactivity distribution was compared in normal mice and nude mice bearing osteogenic sarcoma. The validity of the radiochemical design of mAbs using the metabolizable linkage to achieve target-selective radioactivity localization will be discussed. MATERIALS AND METHODS

Reagents and Chemicals. Na131I and Na125I were obtained from Daiichi Radioisotopes Laboratories (Tokyo)

Metabolizable Linkages for Radiolabeled Antibodies

and Daiichi Kagaku (Tokyo), respectively, and were diluted with 0.1 M PB (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 kindly supplied by Nihon Medi-Physics (Nishinomiya, Japan). The stannyl precursors of HML, 3′-(tri-n-butylstannyl)hippuryl-N-maleoylL-lysine (SHML), MPH, N-(5-maleimidopentyl) 3′-(tri-nbutylstannyl)hippuric acid amide (SMPH), and MIH, maleimidoethyl 3-(tri-n-butylstannyl)hippurate (SMIH), were synthesized as reported previously (25, 26). EMCSBz-EDTA was purchased from Dojindo Laboratories (Kumamoto, Japan). Size-exclusion HPLC and RPHPLC were performed using Cosmosil Diol-300 (7.5 × 600 mm, Nacalai Tesque, Kyoto, Japan) and Cosmosil 5C18-AR (4.6 × 250 mm, Nacalai Tesque) columns, respectively. Size-exclusion HPLC and RP-HPLC columns were equilibrated and eluted with 0.1 M 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 were used as received. Tumor and mAbs. KT005-cloned human osteogenic sarcoma was maintained by serial subcutaneous transplantation in athymic mice. Pieces (1-2 mm3) of tumor tissue (ca. 0.3-0.9 g) at 2 weeks postplantation were used for the in vivo study. Single-cell suspensions from xenografted tumors were used for the in vitro study. The mAb against osteogenic sarcoma (OST7, IgG1), generated by standard hybridoma techniques, was purified by ammonium sulfate precipitation with subsequent protein A affinity chromatography (Pharmacia Biotech Co., Ltd., Tokyo) as reported previously (2, 11, 28). Preparation of Radioiodinated OST7 Conjugates. SHML, SMPH, and SMIH were radioiodinated in the presence of NCS as previously described (11, 25, 26). Briefly, SHML was dissolved in methanol containing 1% acetic acid (0.64 mg/mL), and 22 µL (0.021 µmol) of this solution was mixed with 6.04 µL of NCS in methanol (0.5 mg/mL) in a sealed vial, followed by an addition of Na131I (3 µL). After incubation at room temperature for 45 min, the reaction was quenched with aqueous sodium bisulfite (2.92 µL, 0.72 mg/mL). The radiochemical yields of [131I]HML were determined by TLC developed with a mixture of chloroform/methanol/acetic acid (40:5:2). The solvent was removed by a flow of N2 prior to the subsequent conjugation reaction with OST7. Radioiodination of SHML with Na125I was performed as described above except using Na125I instead of Na131I. Conjugation of [131I]HML with OST7 was performed by reducing the disulfide bonds of OST7, as described previously (2, 11). Briefly, OST7 (250 µL, 5 mg/mL) in well-degassed 0.1 M PB (pH 7.0) containing 2 mM EDTA was allowed to react with a 1000-fold molar excess of 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 Co., Ltd.) equilibrated and eluted with 0.1 M PB (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 (11, 30, 32). The filtrate (90 µL) was then added to a reaction vial containing crude [131I]HML. After gentle agitation of the reaction mixture for 1.5 h at room temperature, 39 µL of iodoacetamide (10 mg/mL) in 0.1 M PB (pH 6.0) was added. The reaction mixture was further incubated for 30 min to alkylate the unreacted thiol groups. [131I]HML-OST7 was subsequently purified by the centrifuged column procedure, equilibrated, and eluted with 0.1 M PBS (pH 6.0). [125I]HMLOST7 was prepared with similar procedures.

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[125I]MIH-OST7 and [125I]MPH-OST7 were also prepared by experimental procedures similar to those used for [125/131I]HML-OST7 except using SMIH or SMPH in place of SHML. Direct radioiodination of OST7 was performed by the chloramine T method (11, 31). To 200 µL aliquots of OST7 (0.25 mg/mL) in 0.3 M PB (pH 7.5) was added 5 µL of Na125I. 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]OST7 was then purified by the centrifuged column procedure. Radiochemical purities of the radioiodinated OST7 conjugates were determined by size-exclusion HPLC, TLC, and CAE. CAE was run at an electrostatic field of 0.8 mA/cm for 45 min in veronal buffer (I ) 0.06, pH 8.6). Preparation of [111In]EMCS-Bz-EDTA-OST7. OST7 was also labeled with 111In using EMCS-Bz-EDTA as the bifunctional chelating agent. EMCS-Bz-EDTA was conjugated to OST7 by reducing the disulfide bonds in OST7 with procedures similar to those used for [131I]HML conjugation, as previously reported (11). EMCS-BzEDTA-OST7 was purified from unreacted low-molecular weight compounds by the centrifuged column procedure using Sephadex G-50 equilibrated and eluted with 20 mM MES-buffered saline (pH 6.0). The number of EMCSBz-EDTA molecules introduced per molecule of OST7 was determined by measuring the thiol groups before and after conjugation reaction using 2,2′-dithiopyridine, as previously described (11, 32). The purified EMCS-Bz-EDTA-OST7 was labeled with 111 In according to the procedure of Brechbiel et al. (33) with slight modifications as previously noted (12, 32). 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 EMCS-Bz-EDTA-OST7 (1 mg/mL) was added, and the reaction solution was kept at room temperature (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 the reaction solution. After incubation for an additional 30 min at room temperature, [111In]EMCS-BzEDTA-OST7 was purified by the centrifuged column procedure using Sephadex G-50 equilibrated with 0.1 M PBS (pH 6.0). The radiochemical purity of [111In]EMCSBz-EDTA-OST7 was determined by CAE and sizeexclusion HPLC. Immunoreactivity Measurement. The immunoreactivity of the four radiolabeled OST7 conjugates was determined as described previously (2, 11, 28). Tumor cells (2 × 104 to 1 × 107) suspended in 100 µL of Dulbecco’s PBS were incubated with 100 µL of radiolabeled OST7 in microcentrifugation tubes (5.7 mm × 46 mm) for 2 h at 4 °C. After centrifugation at 10000g for 5 min, the supernatant was discarded, and the radioactivity was determined using a well counter (ARC 2000, Aloka, Tokyo). Stability Estimation of Radiolabeled OST7 Conjugates in Human Serum. Each radiolabeled OST7 was 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 human serum (11). 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 the three radioiodinated OST7 conjugates were determined by PC (no. 50, Advantec Toyo, Tokyo) and TLC. The percentages of the radioactivity released from [111In]EMCS-Bz-EDTA-OST7 were determined by size-

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Scheme 1a

a (a) N-Chlorosuccinimide, Na131/125l, 1% AcOH/MeOH, room temperature, 45 min; (b) 2-mercaptoethanol, 0.1 M PBS/2 mM EDTA (pH 6.0), room temperature, 30 min; (c) 0.1 M PBS/2 mM EDTA (pH 6.0), 1.5 h; (d) iodoacetamide, 0.1 M PB (pH 6.0), room temperature, 30 min.

exclusion HPLC, as reported previously (32). Each value was calculated by dividing the antibody-bound radioactivity at various intervals by the radiochemical purity of the respective freshly prepared 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 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. In Vivo Studies. In vivo studies were performed after simultaneous administration of a mixed solution of [131I]HML-OST7 and [125I]MIH-OST7 or [125I]MPH-OST7. A 3 mL solution of 0.1 M PB (pH 6.0) containing 1.12 mg of unmodified OST7 was mixed with 12 µL each of [131I]HML-OST7 (14.6 µCi) and [125I]MIH-OST7 or [125I]MPH-OST7 (9.3 or 10.0 µCi) prior to administration. For comparison of the radioactivity distribution of [125I]HMLOST7 and [111In]EMCS-Bz-EDTA-OST7, 12 µL (10.9 µCi, 42 µg) of [125I]HML-OST7 and 63 µL (10.5 µCi, 42 µg) of [111In]EMCS-Bz-EDTA-OST7 were added to 3 mL of unmodified OST7 (1.12 mg) prior to administration. Blood clearance of radioactivity after intravenous administration of each mixture in 6-week-old male ddY mice (34) was determined. Groups of five mice, each receiving 100 µL of the radiolabeled OST7 conjugate solution, were sacrificed 1, 3, 6, 24, 48, 72, and 96 h postinjection. Organs of interest were removed and weighed, and the radioactivity was determined with a well counter (ARC 2000). A window from 29 to 97 keV was used for counting 125I while one from 280 to 440 keV for 131I and from 168 to 568 keV for 111In. Correlation factors for eliminating crossover of 131I or 111In activity into 125I were determined by counting the 131I or 111In standard in each window. The crossover of 125I into the 131I or 111In channels was negligible. Samples of urine and feces were collected for 96 h, and the radioactivity was determined. Twenty-four hours after injection of [125I]HML-OST7, urine samples were collected and analyzed by TLC as described above. The urine samples were also analyzed by RP-HPLC after filtration through a 10 kDa cutoff ultrafiltration membrane (Microcon 10, Amicon Grace, Tokyo). In RP-HPLC analysis, free iodine, m-iodohippuric acid, and m-iodobenzoic acid had retention times of 2.9, 9.4, and 30.8 min, respectively.

Athymic mice bearing osteogenic sarcoma were also treated intravenously with 100 µL of a mixed solution of [131I]HML-OST7 and [125I]MIH-OST7, [131I]HML-OST7 and [125I]MPH-OST7, and [125I]HML-OST7 and [111In]EMCS-Bz-EDTA-OST7, prepared as described above. The animals were sacrificed 24 and 48 h postinjection (five mice in each group). 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 the unpaired t test for in vitro studies and the 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 OST7 Conjugates. Radioiodination of SHML was performed in 1% AcOH/ MeOH in the presence of NCS as an oxidant (Scheme 1). After reaction for 45 min at room temperature, [125I]HML and [131I]HML were obtained with radiochemical yields of 80.2 and 78.9%, respectively, as determined by TLC. Similar procedures were employed for the radioiodination of SMIH and SMPH, which provided [125I]MIH and [125I]MPH with radiochemical yields of 79.8 and 88.7%, respectively. All the radioiodinated compounds were treated with sodium bisulfite to quench any residual NCS, preventing the iodine species from reacting with tyrosine residues of the antibody molecule. Neutralization of NCS also prevented exposure of the protein to the oxidant. Conjugation of [125/131I]HML with OST7 was performed by reactions of thiolated antibodies with the maleimide group of [125/131I]HML. 2-ME reduction of OST7 exposed 6.2 thiol groups per molecule of OST7, and subsequent conjugation reactions with [125/131I]HML were performed at a maleimide-to-thiol molar ratio of 1:1. After the conjugation reaction, the unreacted thiol groups were capped with iodoacetamide. The reaction steps are summarized in Scheme 1. The radiochemical yield of [131I]HML-OST7 was 46.8%, as determined by CAE. After purification by the centrifuged column procedure, [131I]HML-OST7 was obtained with a radiochemical purity and specific activity of 96.0% and 348 µCi/mg,

Metabolizable Linkages for Radiolabeled Antibodies

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Figure 2. Size-exclusion HPLC profile of the radioactivity of [131I]HML-OST7. More than 97% of the radioactivity was eluted at a retention time identical to that of unmodified OST7. Under these conditions, thyroglobulin (670 000), OST7 (IgG), bovine serum albumin (68 000), and cytochrome c (13 000) were eluted at retention times of 11.4, 15.4, 16.8, and 21.0 min, respectively.

respectively, for subsequent studies. With similar procedures, [125I]HML-OST7, [125I]MIH-OST7, and [125I]MPH-OST7 were obtained with radiochemical yields of 67.1, 72.3, and 81.9%, respectively. The radiochemical purity and specific activity were 95.1% and 261 µCi/mg, 98.7% and 223 µCi/mg, and 99.4% and 238 µCi/mg for [125I]HML-OST7, [125I]MIH-OST7, and [125I]MPHOST7, respectively. Direct radioiodination of OST7 with Na125I produced [125I]OST7 with a radiochemical purity and specific activity of 97.3% and 74 µCi/mg, respectively. The EMCS-Bz-EDTA-OST7 conjugate was prepared with maleimide-thiol chemistry using the maleimide group of EMCS-Bz-EDTA and the thiol groups of OST7, generated by reducing the disulfide bonds. The number of EMCS-Bz-EDTA molecules attached per molecule of OST7 was 3.7, as determined by measuring the thiol groups before and after the conjugation reaction. After the centrifuged column procedure, [111In]EMCS-Bz-EDTAOST7 was obtained with a radiochemical purity and specific activity of 91.9% and 250 µCi/mg, respectively, as determined by CAE. Figure 2 shows the size-exclusion HPLC profile of [131I]HML-OST7. More than 97% of the radioactivity was observed in fractions similar to those of unmodified OST7. Similar results were observed with all the radiolabeled OST7 conjugates used in this study (data not shown). Serum Stability of Radiolabeled OST7 Conjugates. Table 1 shows the percentages of the proteinbound radioactivity of the respective radiolabeled OST7 conjugate after incubation in freshly prepared human serum at 37 °C. [131I]HML-OST7, [125I]MPH-OST7, and [111In]EMCS-Bz-EDTA-OST7 released only ca. 2-3% of the initial antibody-bound radioactivity after incubation for 24 h. However, [125I]MIH-OST7 released a significantly higher amount of the radioactivity with incubation time, and half of the initial radioactivity was released from OST7 after incubation for 24 h. The radioactivity released from [125I]MIH-OST7 was an indication of Rf values identical to those of m-iodohippuric acid when analyzed by TLC and PC.

Figure 3. Organ-to-tumor ratios of the radioactivity 6 h (A) and 72 h (B) after injection of [125I]HML-OST7, [131I]HMLOST7, [125I]MIH-OST7, [125I]MPH-OST7, and [111In]EMCSBz-EDTA-OST7 in normal mice. Since no significant differences were observed between [125I]HML-OST7 and [131I]HML-OST7, these results were combined and expressed as the results for [125/131I]HML-OST7.

Immunoreactivity. The binding of [131I]HML-OST7, [125I]HML-OST7, [125I]MIH-OST7, [125I]MPH-OST7, [111In]EMCS-Bz-EDTA-OST7, and [125I]OST7 to KT005 cells was examined as a function of cell number. No significant differences were observed in the cell-bound radioactivity among the six radiolabeled OST7 conjugates (data not shown). Biodistribution Studies in ddY Mice. The time courses of the radioactivity in the blood after intravenous administration of radiolabeled OST7 conjugates in ddY mice are summarized in Table 2. Since [131I]HML-OST7 and [125I]HML-OST7 had similar radioactivity distributions in the three experiments, these results were combined and presented as [125/131I]HML-OST7 in Table 2. While no significant differences were observed in the radioactivity elimination from the blood up to 96 h postinjection among [125/131I]HML-OST7, [125I]MPHOST7, and [111In]EMCS-Bz-EDTA-OST7, [125I]MIHOST7 showed a significantly rapid disappearance of the radioactivity from the blood from 24 h postinjection onward. The liver-to-blood and intestine-to-blood ratios of the radioactivity 6 (A) and 72 h (B) postinjection of

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Table 1. Stabilities of Radiolabeled OST7 Conjugates in Human Serum at 37 °Ca % of protein-bound radioactivity reagent

1

[131I]HML-OST7c

hb

100.00 (0.42) 100.37 (0.61) 98.49 (0.18) ndd

[125I]MPH-OST7c [125I]MIH-OST7c [111In]EMCS-Bz-EDTA-OST7c

3 hb

6 hb

24 hb

99.41 (0.23) 98.15 (3.83) 95.35 (0.16) nd

99.26 (0.23) 99.81 (0.23) 85.72 (1.21) nd

97.45 (0.36) 98.38 (0.35) 49.21 (4.85) 102.45 (3.49)

a Mean (SD) of four experiments for each point. b Incubation time. c Values determined by PC and TLC for radioiodinated OST7 conjugates and by HPLC for the 111In-labeled OST7 conjugate. d nd, not determined.

Table 2. Radioactivity Clearance from the Blood after Intravenous Administration of Radiolabeled OST7 Conjugates in ddY Micea [125/131I]HML-OST7 [125I]MIH-OST7 [125I]MPH-OST7 [111In]EMCS-Bz-EDTA-OST7

1 hb

6 hb

24 hb

48 hb

72 hb

96 hb

25.13 (1.69) 25.83 (3.04) 25.50 (2.15) 24.87 (1.45)

19.70 (2.34) 16.90 (0.96) 21.78 (0.83) 19.41 (1.75)

12.16 (0.95) 8.99c (0.30) 12.85 (1.02) 12.15 (1.31)

9.65 (0.78) 7.24c (0.68) 9.45 (0.74) 9.75 (1.33)

7.71 (0.69) 5.23c (0.44) 7.86 (0.98) 7.94 (1.13)

6.44 (0.53) 4.18c (0.60) 6.45 (0.85) 5.97 (0.22)

a Expressed as the percentage of the injected dose per gram of blood. Mean (SD) for four to ten animals for each point. b Time after administration. cp < 0.0005 compared to that for [131I]HML-OST7.

Table 3. Cumulative Radioactivity Excreted in the Urine and Feces 96 h after Injection of Radiolabeled OST7 Conjugates in ddY Micea [125/131I]HML-OST7 [125I]MIH-OST7 [125I]MPH-OST7 [111In]EMCS-Bz-EDTA-OST7

urine

feces

45.94 (8.66) 58.92 (1.40)b 27.13 (3.93)b 26.94 (4.88)b

2.17 (0.22) 1.91 (0.33) 23.43 (0.91)c 9.83 (2.81)b

a Expressed as the percentage of the injected dose. Mean (SD) for four to ten animals for each point. b p < 0.01 compared to that for [125/131I]HML-OST7. c p < 0.0001 compared to that for [125/131I]HML-OST7.

the four radiolabeled OST7 conjugates in ddY mice are shown in Figure 3. No significant differences in the radioactivity ratios were observed between [131I]HMLOST7 and [125I]MIH-OST7 at either postinjection time point. However, [125I]MPH-OST7 had significantly higher liver-to-blood ratios at 72 h and intestine-to-blood ratios at both 6 and 72 h than [131I]HML-OST7. Significantly higher radioactivity ratios were also observed with [111In]EMCS-Bz-EDTA-OST7 at 6 h for the liver and at 6 and 72 h for the intestine when compared with those of simultaneously administered [125I]HML-OST7. Figure 4 shows the RP-HPLC and TLC radioactivity profiles of urine obtained 24 h after injection of [131I]HML-OST7. RP-HPLC indicated that more than 93% of the radioactivity in the urine was eluted at a retention time identical to that of m-iodohippuric acid. TLC analysis also indicated a single radioactivity peak (>98%) at an Rf value identical to that of m-iodohippuric acid. Table 3 summarizes the cumulative radioactivity excreted in the urine and feces 96 h after injection of the five radiolabeled OST7 conjugates. [125I]MIH-OST7 showed the fastest excretion rate of radioactivity from the body. While both [125/131I]HML-OST7 and [125I]MIH-OST7 showed more than 95% excreted radioactivity in the urine, [125I]MPH-OST7 excreted an almost equal amount of radioactivity in urine and feces. [111In]EMCS-Bz-EDTA-OST7 exhibited the slowest excretion rate of radioactivity from the body. Biodistribution Studies in Nude Mice. Table 4 shows the biodistribution of radioactivity after simultaneous administration of [131I]HML-OST7 and [125I]MIHOST7 in athymic mice bearing osteogenic sarcoma. Although similar radioactivity levels in the tumor were observed with both radioiodinated OST7 conjugates 24 h postinjection, [125I]MIH-OST7 indicated a significant

Figure 4. Radioactivity profiles of murine urine 24 h after injection of [131I]HML-OST7 on RP-HPLC (A) and TLC (B). The major radioactivity (>93% for RP-HPLC and >98% for TLC) was detected at a retention time and Rf value similar to those of m-iodohippuric acid.

decrease in the radioactivity levels in the tumor 48 h postinjection. Although [125I]MIH-OST7 indicated significantly higher tumor-to-liver and tumor-to-intestine ratios of radioactivity at both postinjection time points, no significant differences were observed between the two

Metabolizable Linkages for Radiolabeled Antibodies

Bioconjugate Chem., Vol. 9, No. 4, 1998 503

Table 4. Comparative Radioactivity Localization after Simultaneous Administration of [131I]HML-OST7 and [125I]MIH-OST7 in Athymic Mice Bearing Osteogenic Sarcomaa % ID/gram tumor blood

time (h) [131I]HML-OST7

24 48 24 48

[125I]MIH-OST7

32.65 (1.54) 30.33 (1.46) 32.41 (2.99) 23.13d (3.57)

liver

9.39 (1.50) 6.20 (1.39) 8.36 (1.40) 3.46c (0.98)

tumor-to-organ intestine

12.93 (2.38) 28.76 (4.63) 15.89b (3.27) 46.52c (10.35)

organ-to-blood liver intestine

41.04 (8.72) 65.40 (9.25) 52.73b (13.78) 132.96c (31.96)

0.23 (0.04) 0.23 (0.02) 0.21 (0.04) 0.23 (0.04)

0.10 (0.01) 0.09 (0.01) 0.10 (0.01) 0.10 (0.01)

a Values are the means (SD) of nine animals for each point. Mean weights (SD) of mice and tumors in grams: 19.62 (1.26) and 0.46 (0.11) at 24 h and 18.92 (0.61) and 0.85 (0.07) at 48 h. b p < 0.05 compared to that for [131I]HML-OST7. c p < 0.01 compared to that for [131I]HML-OST7. d p < 0.005 compared to that for [131I]HML-OST7.

Table 5. Comparative Radioactivity Localization after Simultaneous Administration of [131I]HML-OST7 and [125I]MPH-OST7 in Athymic Mice Bearing Osteogenic Sarcomaa time (h) [131I]HML-OST7

24 48

[125I]MPH-OST7

24 48

% ID/gram tumor blood 33.72 (3.21) 31.31 (5.40) 32.50 (2.75) 29.75 (3.83)

9.37 (0.71) 8.09 (0.23) 8.52 (0.56) 7.53 (0.27)

tumor-to-organ liver intestine 15.86 (1.24) 24.48 (5.85) 15.75 (3.39) 24.52 (8.37)

60.10 (4.63) 72.42 (24.22) 38.10b (4.63) 32.43b (9.26)

a Values are the means (SD) of five animals for each point. Mean weights (SD) of mice and tumors in grams: 20.69 (0.77) and 0.54 (0.22) at 24 h and 20.46 (1.92) and 0.51 (0.21) at 48 h, respectively. b p < 0.005 compared to that for [131I]HML-OST7.

radioiodinated OST7 conjugates when the liver-to-blood and intestine-to-blood ratios of the radioactivity were calculated. The biodistributions of radioactivity after simultaneous administration of [131I]HML-OST7 and [125I]MPH-OST7 in athymic mice bearing osteogenic sarcoma are summarized in Table 5. Both radioiodinated OST7 conjugates showed no changes in radioactivity levels in the tumor between the two postinjection time points. No significant differences were observed in the radioactivity levels in the tumor and in the blood when the two radioiodinated OST7 conjugates were compared 24 and 48 h postinjection. However, while [131I]HML-OST7 and [125I]MPH-OST7 had similar tumor-to-liver ratios of radioactivity, significantly higher tumor-to-intestine ratios were achieved by [131I]HML-OST7 at both postinjection time points. The biodistributions of radioactivity after simultaneous administration of [125I]HML-OST7 and [111In]EMCS-BzEDTA-OST7 in athymic mice are shown in Table 6. [125I]HML-OST7 and [111In]EMCS-Bz-EDTA-OST7 had similar radioactivity levels in the tumor and blood both

24 and 48 h postinjection. However, [125I]HML-OST7 had significantly higher tumor-to-liver ratios of radioactivity both 24 and 48 h postinjection and tumor-tointestine ratios 48 h postinjection when compared with [111In]EMCS-Bz-EDTA-OST7. DISCUSSION

All the radiolabeled OST7 conjugates were prepared using similar conjugation chemistry. All exhibited similar radiochromatograms on size-exclusion HPLC (Figure 2) and similar affinities for KT005 cells. In addition, high stability against in vivo deiodination or transchelation of all the radiolabeled OST7 conjugates was well demonstrated in this study and in previous studies (11, 12, 14, 25-27, 32, 35). Thus, the differences in the localization of the radioactivity from the four radiolabeled OST7 conjugates in vivo could be primarily attributed to the differences in stability of the metabolizable linkage of the respective radiolabeled OST7 and the in vivo behavior of radiometabolites derived from each reagent. The plasma-labile characteristic of the ester bond in MIH (Table 1) was reflected in the biodistribution studies in normal mice, and the fastest elimination rate of radioactivity from the blood (Table 2) and the highest excretion of the radioactivity from the body (Table 3) were observed with [125I]MIH-OST7. Despite this, however, no significant differences were observed in the organ-toblood ratios of the radioactivity between [131I]HMLOST7 and [125I]MIH-OST7 (Figure 3), indicating that [131I]HML-OST7 liberated m-iodohippuric acid after lysosomal proteolysis in nontarget tissues at a rate similar to that of [125I]MIH-OST7. This was supported by the results of the urine analyses where the radioactivity was excreted as m-iodohippuric acid after administration of [131I]HML-OST7 (Figure 4). Since similar radioactivity levels in the blood were observed after simultaneous administration of [131I]HML-OST7 and [125I]MPH-OST7 (Table 2), similar amounts of radiolabeled OST7 would be delivered to the liver. Thus, significantly higher radioactivity levels in

Table 6. Comparative Radioactivity Localization after Simultaneous Administration of [131I]HML-OST7 and [111In]EMCS-Bz-EDTA-OST7 in Athymic Mice Bearing Osteogenic Sarcomaa % ID/gram time (h) [131I]HML-OST7

24 48

[111In]EMCS-Bz-EDTA-OST7

24 48

tumor-to-organ

tumor

blood

liver

intestine

37.14 (2.94) 29.61 (9.51) 39.53 (2.93) 33.39 (7.06)

10.07 (1.70) 7.19 (1.43) 8.15 (0.61) 5.47 (1.02)

12.59 (2.37) 32.82 (14.88) 3.86c (1.09) 8.41c (3.09)

54.64 (5.80) 82.14 (34.38) 54.21 (6.17) 75.68b (10.06)

a Values are the means (SD) of five animals for each point. Mean weights (SD) of mice and tumors in grams: 20.44 (1.41) and 0.37 (0.10) at 24 h and 20.88 (0.52) and 0.47 (0.25) at 48 h, respectively. b p < 0.05 compared to that for [131I]HML-OST7. c p < 0.005 compared to that for [131I]HML-OST7.

504 Bioconjugate Chem., Vol. 9, No. 4, 1998

these tissues after administration of [125I]MPH-OST7 (Figure 3) would reflect the slower hepatic elimination rate of the radiometabolites derived from MPH and subsequent hepatobiliary excretion, as also observed in a previous study using NGA (25). Similarly, the slowest hepatic elimination rate of the radiometabolites derived from [111In]EMCS-Bz-EDTA-OST7 would be responsible for the highest liver-to-blood ratios of the radioactivity of this radiolabeled OST7. This was supported by the lowest radioactivity excretion levels of this radiolabeled OST7 (Table 3). These findings using OST7 were consistent with those of previous studies using NGA as a model polypeptide, reinforcing the fact that in vivo behavior of radiometabolites generated after lysosomal proteolysis of radiolabeled mAbs plays a critical role in the hepatic radioactivity levels. These findings also indicated that, when applied to mAbs, HML provided rapid and selective release of m-iodohippuric acid after accumulation in nontarget tissues such as the liver, while providing a stable attachment for m-iodohippuric acid with the mAb in plasma. To further estimate the validity of the radiochemical design of mAbs with metabolizable linkages, two radiolabeled OST7 conjugates that liberate m-iodohippuric acid as the sole radiometabolite after lysosomal proteolysis at a similar rate, [131I]HML-OST7 and [125I]MIHOST7, were simultaneously administered to nude mice bearing osteogenic sarcoma. Similar radioactivity levels in the tumor 24 h after injection of the two radiolabeled OST7 conjugates would be attributable to the similar radioactivity levels in the blood at this postinjection time and the similar affinities for the tumor cells of the two radioiodinated OST7 conjugates. The significantly lowered radioactivity levels in the blood 48 h after injection of [125I]MIH-OST7 would be caused by the enzymatic and chemical hydrolysis of the ester bond in the blood, followed by the urinary excretion of the resulting miodohippuric acid. On the other hand, the formation of antigen-antibody complexes on the tumor cell surface provides steric hindrance to esterase access, which stabilized the ester bond of [125I]MIH-OST7 (11). Thus, while chemical hydrolysis of the ester bond in [125I]MIHOST7 would be predominantly responsible for the elimination of radioactivity from the tumor, both chemical and enzymatic hydrolyses takes place in the blood, which enhances the target-to-nontarget ratios of the radioactivity with time after injection (Table 4). Since the peptide bond of [131I]HML-OST7 was stable both in the blood and on the tumor cell surface, neither accelerated clearance of the radioactivity from the blood nor decreased radioactivity levels in the tumor were observed. Although [125I]MIH-OST7 provided significantly higher tumor-to-nontarget ratios of the radioactivity, these results would be most likely caused by the presence of large tumor masses compared to their small distribution volume in the animal model. When the much lower and delayed accumulation of the radioactivity of radiolabeled mAbs in clinical studies is considered (36-39), the plasma-labile characteristics of the ester bond in MIH may cause a significant decrease in the radioactivity levels in the tumor in clinical studies. Thus, the animal model may have underestimated the plasma-labile characteristics of the ester bond in MIH in the radioactivity delivery to the target by the mAb. Similar nontargetto-blood ratios of the radioactivity after administration of [131I]HML-OST7 and [125I]MIH-OST7 in normal and nude mice also suggested that the higher tumor-tonontarget ratios of the radioactivity with [125I]MIH-

Arano et al.

OST7 are characteristic of the animal model (Figure 3 and Table 4). The ability of HML to provide target-selective radioactivity localization using mAbs as vehicles was clearly demonstrated in the comparative biodistribution studies of [125/131I]HML-OST7 with [125I]MPH-OST7 or [111In]EMCS-Bz-EDTA-OST7 (Tables 5 and 6). Similar radioactivity levels in the blood and the tumor both 24 and 48 h after injection of the three radiolabeled OST7 conjugates coincided well with the similar plasma stabilities and tumor cell affinities of the three radiolabeled OST7 conjugates. Significantly higher radioactivity levels in the intestine of [125I]MPH-OST7 and in the liver and intestine of [111In]EMCS-Bz-EDTA-OST7 reflected the in vivo behavior of radiometabolites derived from the respective reagent. These findings indicated that the radiochemical design of mAb conjugates that provides a stable attachment for the radiolabel in plasma while facilitating rapid and selective release of the radiometabolite of urinary excretion would be useful in achieving target-selective radioactivity localization using mAbs as vehicles. Recently, efforts have been directed toward increasing the accumulation of radiolabeled mAbs by hyperthermia or biological response modifiers (40-43). Antibody fragments and mAbs that recognize the antigens present on the vascular wall in the tumoral tissues offer promise for achieving target-selective radioactivity localization within short postinjection intervals (18, 20-24). Since the radiochemical design of mAb conjugates using metabolizable linkages is complementary to these approaches, combinations of these different approaches would render the radiolabeled mAbs attractive radiopharmaceuticals for both diagnostic and therapeutic nuclear medicine. In conclusion, the peptide bond of HML-conjugated OST7 remained stable in plasma but rapidly liberated m-iodohippuric acid after lysosomal proteolysis in nontarget tissues. As a result, HML-conjugated OST7 decreased the radioactivity levels in nontarget tissues without impairing those in the target delivered by the mAb. These findings strongly supported the validity of the radiochemical design of antibodies using metabolizable linkages that provide a stable attachment of radiolabels with mAbs in plasma while facilitating selective release of radiometabolites of rapid urinary excretion. The well-correlated hepatic radioactivity levels between radiolabeled mAbs and radiolabeled NGAs also indicated that the biological method using NGA as the model polypeptide would be highly useful in estimating newly designed radiolabeling reagents for reducing hepatic radioactivity levels of radiolabeled mAbs. ACKNOWLEDGMENT

We are grateful to Dr. I. Yomoda of Daiichi Radioisotope Laboratories (Tokyo) for providing Na131I. We also thank Nihon Medi-Physics for their kind gift of 111InCl3. This study was supported in part by Grants-in-Aid for Developing Scientific Research (08557135 and 09672279) from the Ministry of Education, Science, Culture and Sports of Japan. LITERATURE CITED (1) 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.

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