(HML)-Conjugated Fab Fragments - American Chemical Society

In this study, renal metabolism of HML-conjugated Fab fragments prepared by different ..... Groups of five mice, each receiving 20 μg of Fab fragment...
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Bioconjugate Chem. 2001, 12, 178−185

Renal Metabolism of 3′-Iodohippuryl NE-Maleoyl-L-lysine (HML)-Conjugated Fab Fragments Yasushi Fujioka,† Yasushi Arano,*,‡ Masahiro Ono,† Tomoya Uehara,‡ Kazuma Ogawa,† Shinji Namba,† Tsuneo Saga,§ Yuji Nakamoto,§ Takahiro Mukai,§ Junji Konishi,§ and Hideo Saji† Department of Patho-Functional Bioanalysis, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Laboratory of Radiopharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, and Department of Nuclear Medicine and Diagnostic Imaging, Graduate School of Medicine, Kyoto University, 54 Shogoin-kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan . Received June 13, 2000; Revised Manuscript Received October 5, 2000

Renal localization of radiolabeled antibody fragments constitutes a problem in targeted imaging and radiotherapy. Recently, we reported use of a novel radioiodination reagent, 3′-[131I]iodohippuryl Nmaleoyl-L-lysine (HML), that liberates m-iodohippuric acid before antibody fragments are incorporated into renal cells. In mice, HML-conjugated Fab demonstrated low renal radioactivity levels from early postinjection times. In this study, renal metabolism of HML-conjugated Fab fragments prepared by different thiolation chemistries and by direct radioiodination were investigated to determine the mechanisms responsible for the low renal radioactivity levels. Fab fragments were thiolated by 2-iminothiolane modification or by reduction of disulfide bonds in the Fab fragments, followed by conjugation with radioiodinated HML to prepare [131I]HML-IT-Fab and [125I]HML-Fab, respectively. In biodistribution studies in mice, both [131I]HML-IT-Fab and [125I]HML-Fab demonstrated significantly lower renal radioactivity levels than those of [125I]Fab. In subcellular distribution studies, [125I]Fab showed migration of radioactivity from the membrane to the lysosomal fraction of the renal cells from 10 to 30 min postinjection. On the other hand, the majority of the radioactivity was detected only in the membrane fraction at the same time points after injection of both [131I]HML-IT-Fab and [125I]HML-Fab. In metabolic studies, while [125I]Fab remained intact at 10 min postinjection, both HMLconjugated Fab fragments generated m-iodohippuric acid as a radiometabolite at the same postinjection time. [131I]HML-IT-Fab registered two radiometabolites (intact [131I]HML-IT-Fab and m-iodohippuric acid), whereas additional radiometabolites were observed with [125I]HML-Fab. This suggested that metabolism of both HML-conjugated Fab fragments would occur in the membrane fractions of the renal cells. The findings of this study reinforced our previous hypothesis that radiochemical design of antibody fragments that liberate radiometabolites that are excreted into the urine by the action of brush border enzymes would constitute a useful strategy to reduce renal radioactivity levels from early postinjection times.

INTRODUCTION

Antibody fragments such as Fab and single-chain Fv fragments show rapid targeting to tumor and uniform distribution in tumor tissues (1). Such properties render antibody fragments useful to deliver radioactivity for both targeted imaging and radiotherapy. However, radiolabeled antibody fragments show persistent localization of radioactivity in the kidneys, which impairs diagnostic accuracy in the kidney region and limits therapeutic potential (2-8). Thus, radiolabeled antibody fragments would become much more useful in diagnosis and targeted radiotherapy if the renal radioactivity could be reduced without impairing the radioactivity levels in the target. Recent studies indicated that the persistent localization of the radioactivity after administration of radiolabeled antibody fragments and peptides was attributable * To whom correspondence should be addressed. Phone: 81-43-290-3024. Fax: 81-43-290-3025. E-mail: arano@ p.chiba-u.ac.jp. † Department of Patho-Functional Bioanalysis. ‡ Laboratory of Radiopharmaceutical Chemistry. § Department of Nuclear Medicine and Diagnostic Imaging.

Figure 1. Chemical structure of [131I]HML.

to slow elimination rates of radiometabolites generated after lysosomal proteolysis of the parental Fab fragments in renal cells (7, 9-11). There have been a number of attempts to facilitate elimination of radiometabolites generated after lysosomal proteolysis of glomerularly filtered antibody fragments through the use of cleavable linkages between antibody fragments and radiolabeled compounds (12-14) (Figure 2A). Although the rationale behind the radiochemical design is well supported by recent metabolic studies as cited above, this approach may also impair target radioactivity levels when applied

10.1021/bc000066j CCC: $20.00 © 2001 American Chemical Society Published on Web 01/10/2001

Renal Metabolism of HML-Conjugated Fab Fragments

Figure 2. (A) Potential mechanism of renal radioactivity localization of radiolabeled Fab fragments. Slow elimination rates of radiometabolites (9) from the lysosomal compartment would be responsible for the long residence time of the radioactivity in the kidney. (B) Proposed mechanism of renal metabolism of [131I]HML-IT-Fab. [131I]HML-IT-Fab would release m-[131I]iodohippuric acid (b) before the antibody fragments are incorporated into the renal cells by the action of the brush border enzymes.

to antibody fragments or peptides that are incorporated into target cells. We recently reported that radioiodinated Fab fragments labeled with 3′-[131I]iodohippuryl N-maleoyl-Llysine ([131I]HML, Figure 1) demonstrated markedly low renal radioactivity levels with kidney-to-blood ratios of 1 from 10 min to 1 h after injection into mice due to rapid release of m-[131I]iodohippuric acid. In addition, HMLconjugated Fab did not impair the radioactivity levels in the tumor when compared with directly radioiodinated Fab in nude mice model (15, 16). On the other hand, radioiodinated Fab via direct radioiodination reached its peak kidney-to-blood ratios of 7.3 at 1 h postinjection. Since the two simultaneously administered radioiodinated Fab fragments showed similar radioactivity levels in the blood, we hypothesized that [131I]HML-conjugated Fab fragments would release m-[131I]iodohippuric acid before the antibody fragments were incorporated into the renal cells (Figure 2B). That is, the glycyl-lysine sequence in HML would be cleaved by the action of the brush border enzyme carboxypeptidase M (17) present on the lumen of renal tubules (18, 19). Since high-level and persistent localization of the radioactivity is also observed with radiolabeled peptides, further investigation of the mechanism responsible for the low renal radioactivity levels of [131I]HML-conjugated Fab fragments would provide an insight for designing radiolabeled antibody fragments and peptides much more useful for both targeted imaging and radiotherapy. In the present study, we investigated renal metabolism of [131I]HML-conjugated Fab fragments. Since esterasemediated cleavage of the ester bonds is affected by the distance between the parental protein and the substrate

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Figure 3. Preparation of radioiodinated Fab fragments. (A) [131I]HML-IT-Fab, (B) [125I]HML-Fab, (C) [125I]Fab. Reagents: (a) 2-IT, 0.16 M borate buffer/2 mM EDTA (pH 8.0); (b) [131I]HML, 0.1 M phosphate buffer/2 mM EDTA (pH 6.0); (c) iodoacetamide, 0.1 M phosphate buffer/2 mM EDTA (pH 6.0); (d) 2-ME, 0.1 M phosphate buffer/2 mM EDTA (pH 7.0); (e) chloramine T, Na[125I]I, 0.3 M phosphate buffer (pH 7.3).

(20, 21), radioiodinated HML was conjugated to Fab fragments via different thiolation chemistries to locate the glycyl-lysine sequence in HML at a position distal or close to the parental Fab fragments; Fab fragments were thiolated by reacting 2-iminothiolane (2-IT) with amine residues of the protein ([131I]HML-IT-Fab, Figure 3A) or by reducing the disulfide bonds with 2-mercaptoethanol (2-ME) ([125I]HML-Fab, Figure 3B). Fab fragments were also directly radioiodinated by the chloramine T method ([125I]Fab, Figure 3C) as a control. Subcellular localization and chemical forms of the radioactivity remaining in the kidney at early time points after injection of the three radioiodinated Fab fragments were investigated and the mechanism of renal metabolism of each radioiodinated Fab fragment will be discussed. The findings of this study provided further evidence that the low renal radioactivity levels of HML-conjugated Fab fragments are due to rapid release of m-iodohippuric acid before incorporation of the fragments into the renal cells. MATERIALS AND METHODS

Reagents and Chemicals. Na[131I]I and Na[125I]I were obtained from Daiichi Radioisotope Laboratories (Tokyo, Japan) and Daiichi Kagaku (Tokyo), respectively, and were diluted with 0.1 M phosphate buffer (pH 7.4) to 37 MBq (1 mCi)/2-10 µL. Size-exclusion highperformance liquid chromatography (SE-HPLC) was performed with a Cosmosil Diol-120 column (7.5 × 600 mm, Nacalai Tesque, Kyoto, Japan) eluted with 0.1 M phosphate buffer (pH 6.8) at a flow rate of 1 mL/min. Reversed-phase HPLC (RP-HPLC) was performed with a Cosmosil 5C18-AR-300 column (4.6 × 150 mm, Nacalai Tesque) eluted with a mixture of 0.1% aqueous phospho-

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ric acid and acetonitrile (3:1) at a flow rate of 1 mL/min. The eluent was collected with a fraction collector (RediFrac; Pharmacia Biotech, Tokyo) at 30-s intervals, and the radioactivity levels in each fraction (500 µL) were determined with an auto well counter (ARC-2000; Aloka, Tokyo). TLC analyses were performed with silica plates (Merck Art 5553). To facilitate collection of urine and feces after administration of radiolabeled Fab, mice were housed in metabolic cages (Metabolica, MM type; Sugiyama-Gen Iriki Co. Ltd., Tokyo). Other reagents were of reagent grade and were used as received. Monoclonal Antibody (mAb). The mAb against osteogenic sarcoma (OST7, IgG1), generated by the standard hybridoma technique, was purified by ammonium sulfate precipitation with subsequent protein A affinity chromatography (Pharmacia Biotech Co. Ltd., Tokyo), as reported previously (22). The Fab fragment was prepared by the standard procedure using papain (23). Preparation of Radioiodinated Fab Fragments. [131/125I]HML was prepared as described previously (21). Conjugation of [131/125I]HML with the Fab fragment of OST7 was performed as illustrated in Figure 3. [131I]HML-IT-Fab was prepared by treating the Fab fragment with 2-IT, followed by conjugation reaction between the thiol groups of Fab and the maleimide groups of [131I]HML (Figure 3A). Briefly, a solution of Fab (200 µL, 2 mg/mL) in well-degassed 0.16 M borate buffer (pH 8.0) containing 2 mM EDTA was allowed to react with 7.2 µL of 2-IT solution (1 mg/mL) prepared in the same buffer. After gentle agitation of the reaction mixture for 60 min at room temperature, excess 2-IT was removed by a centrifuged column procedure (24) using Sephadex G-50 (Pharmacia Biotech Co. Ltd.) equilibrated and eluted with 0.1 M phosphate buffer (pH 6.0) containing 2 mM EDTA. Aliquots of this mixture were sampled for estimation of the number of thiol groups with 2,2′dipyridyl disufide (25). The filtrate (100 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, 14.8 µL of iodoacetamide (10 mg/mL) in 0.1 M phosphate buffer (pH 6.0) containing 2 mM EDTA was added. The reaction mixture was further incubated for 30 min to alkylate the unreacted thiol groups. [131I]HMLIT-Fab was subsequently purified by the centrifuged column procedure, equilibrated and eluted with 0.1 M acetate buffer (pH 6.0). [125I]HML-Fab was prepared by reducing disulfide bonds in the Fab fragment with 2-ME, followed by conjugation between Fab and [125I]HML (Figure 3B). Briefly, a solution of Fab (200 µL, 2 mg/mL) in welldegassed 0.1 M phosphate buffer (pH 7.0) containing 2 mM EDTA was allowed to react with 5.6 µL of 10-fold diluted 2-ME solution using the same buffer and stirred gently for 60 min at room temperature. The experimental procedures for preparation of [125I]HML-Fab after introduction of thiol groups were similar to those used for [131I]HML-IT-Fab except that [125I]HML was used in place of [131I]HML. Direct radiodination of Fab was performed by the chloramine T method (Figure 3C) (15). To 100 µL aliquots of Fab (2.5 mg/mL) in 0.3 M phosphate buffer (pH 7.3) was added 1 µL of Na[125I]I. Chloramine T (0.1 mg/mL, 12.5 µL), freshly prepared in the same buffer, was then added. After incubation of the mixture for 5 min at room temperature, [125I]Fab was purified by the centrifuged column procedure, as described above.

Fujioka et al.

Radiochemical purities of the radioiodinated Fab fragments were determined by SE-HPLC and TLC developed with a mixture of methanol and water (4:1). Biodistribution of Radioiodinated Fab Fragments. In vivo studies were performed after simultaneous administration of a mixed solution of [131I]HMLIT-Fab and [125I]HML-Fab or after injection of a solution of [125I]Fab. Three mL of 0.1 M phosphate buffer (pH 7.4) containing 600 µg of unmodified Fab were mixed with 10 µL of each radioiodinated Fab fragment (12-13 µCi) prior to administration. The biodistribution of radioactivity after i.v. administration of each mixture in 6-weekold male ddY mice (27-30 g) (26) was monitored at 10 and 30 min and 1, 3, 6, and 24 h postinjection. Groups of five mice, each receiving 20 µg of Fab fragments, were used for the experiments. 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, whereas a window from 280 to 440 keV was used for 131I. Correlation factors to eliminate crossover of 131I activity into 125I were determined by counting 131I standard in each window. The crossover of 125I into the 131I channel was negligible. To determine the amounts and routes of excretion of radioactivity from the body, mice were housed in metabolic cages for 24 h after administration of the respective preparation. Urine and feces were collected for 6 and 24 h postinjection, and the radioactivities were determined. Subcellular Distribution of the Radioactivity. The subcellular distribution of the radioactivity in the kidney was determined 10 and 30 min after administration of 20 µg of [131I]HML-IT-Fab, [125I]HML-Fab, or [125I]Fab, according to the procedures described previously (27), with slight modifications. At the indicated times, murine kidneys were isolated, minced, and homogenized in 4 vol of ice-cold 0.25 M sucrose buffered with 10 mM phosphate buffer (pH 7.4) with a Dounce homogenizer by hand (20 strokes). This was followed by two final strokes in an icecold Potter-Elvehjem homogenizer with a Teflon pestle at 800 rpm. The resultant 20% homogenate was centrifuged twice for 5 min each time at 340g at 4 °C. The isolated supernatant was layered on top of iso-osmotic (0.25 M sucrose) Percoll (Pharmacia Biotech Co. Ltd.) at a density of 1.08 g/mL. After centrifugation at 20000g (RP 30 rotor, Hitachi Co. Ltd., Tokyo) for 90 min at 4 °C, the gradient (1.02-1.14 g/mL) was collected in 14 fractions before analyses of β-galactosidase (28) and alkaline phosphodiesterase I (29) activities, density, and the radioactivity of each fraction. Radiolabeled Species in the Kidney. The radiolabeled species remaining in the kidney at 10 and 30 min postinjection of each preparation were analyzed according to the procedure described previously (27). Briefly, each 131/125I-labeled Fab (20 µg) was administered intravenously into mice. At 10 and 30 min postinjection, both kidneys were perfused in situ with cold 0.1 M Tris-citrate buffer (pH 6.5) containing 0.15 M NaCl, 0.02% sodium azide, 1 TIU/mL aprotinin, 2 mM benzamidine-HCl, 2 mM iodoacetamide, 1 mM phenylmethanesulfonyl fluoride, and 5 mM diisopropyl fluorophosphate before being removed. Each tissue sample was placed in a test tube and subjected to three cycles of freezing (dry ice-acetone bath) and thawing. After addition of 5 vol of the same buffer containing an additional 35 mM β-octyl-glucoside, the samples were homogenized with a Polytron homogenizer (PT10-35, Kinematica GmbH Littau, Switzerland) at full speed with three consecutive 30-s bursts on ice before centrifugation at 48000g for 20 min at 4 °C (Himac CS-120 Centrifuge, Hitachi Co. Ltd.). The supernatant

Renal Metabolism of HML-Conjugated Fab Fragments

was separated from the pellet, and the radioactivity was counted. The kidney supernatant was analyzed immediately by SE-HPLC after filtration through a polycarbonate membrane with a pore diameter of 0.45 µm (Nacalai Tesque). Each sample was also analyzed immediately by RP-HPLC and TLC after ultrafiltration with a 10 kDa cutoff membrane (Microcon-10, Amicon Grace, Tokyo). TLC plates were developed with a mixture of chloroform, methanol, and water (15:8:1). Under these conditions, protein-bound radioactivity, free iodine, miodohippuric acid, and m-iodobenzoic acid had Rf values of 0, 0.1, 0.45, and 0.65, respectively. Radiolabeled Species in the Urine. The radiolabeled species excreted in the urine for 6 h postinjection of each 131/125I-labeled Fab were analyzed by SE-HPLC after filtration through a polycarbonate membrane with a pore diameter of 0.45 µm (Nacalai Tesque). After ultrafiltration with a 10 kDa cutoff membrane (Microcon10), the urine samples were also analyzed by RP-HPLC and TLC developed with a mixture of chloroform, methanol, and water (15:8:1) for [131I]HML-IT-Fab and [125I]HML-Fab and by TLC developed with (a) a mixture of methanol and acetic acid (100:1) or (b) a mixture of methanol and 10% ammonium acetate (10:1) for [125I]Fab (30). In point a, monoiodotyrosine and free iodine had Rf values of 0.5 and 0.75, whereas in point b, Rf values were 0.55 and 0.80, respectively. Radiolabeled Species in the Plasma. Blood samples were taken at 10 and 30 min after injection of [131I]HMLIT-Fab, [125I]HML-Fab, or [125I]Fab. Each sample was centrifuged at 800g for 15 min at 4 °C. The supernatant of each sample was analyzed by SE-HPLC after filtration through a polycarbonate membrane with a pore diameter of 0.45 µm (Nacalai Tesque). RESULTS

Preparation of Radioiodinated Fab Fragments. Treatment of Fab fragments with 2-IT or 2-ME introduced 3.65 or 3.67 thiol groups per molecule of Fab, respectively, as determined by 2,2′-dipyridyl disulfide. Conjugation of [131/125I]HML with Fab fragments was performed by reacting the thiolated Fab fragments with maleimide groups of [131/125I]HML. After purification by the centrifuged column procedure, [131I]HML-IT-Fab was obtained with a radiochemical purity and specific activity of 98.5% and 62.5 µCi/mg, respectively, as determined by SE-HPLC. Radiochemical purities and specific activities were 99.3% and 192 µCi/mg for [125I]HML-Fab, and 94.9% and 640 µCi/mg for [125I]Fab, respectively. Biodistribution of Radioiodinated Fab Fragments. Two independent biodistribution studies were performed to achieve the results shown in Figure 4. One was performed after simultaneous administration of a mixed solution of [131I]HML-IT-Fab and [125I]HML-Fab and the other was performed after injection of directly radioiodinated Fab ([125I]Fab). [125I]Fab showed the lowest radioactivity levels in the blood among the three, whereas the two HML-conjugated Fab fragments registered similar radioactivity levels. In the kidney, the two HMLconjugated Fab fragments showed much lower radioactivity levels than those of [125I]Fab. Although [125I]Fab showed the highest radioactivity levels (65% injected dose per gram; ID/g) in the kidney at 30 min postinjection, [131I]HML-IT-Fab and [125I]HML-Fab had no radioactivity peaks. [125I]HML-Fab demonstrated significantly higher radioactivity levels than those of [131I]HML-IT-Fab in the kidney at 10 and 30 min postinjection. Notable differ-

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Figure 4. Biodistributions of radioactivity in mice after injection of [131I]HML-IT-Fab (b), [125I]HML-Fab (O), and [125I]Fab (2). Significance was determined by unpaired Student’s t-test [(*) p < 0.01; (**) p < 0.05 compared to [131I]HML-ITFab].

ences were not observed in other tissues, except for the uptake in the stomach observed with [125I]Fab (data not shown). Subcellular Distribution of the Radioactivity. Percoll density gradient centrifugation profiles of the radioactivity at 10 and 30 min after injection of [131I]HML-IT-Fab, [125I]HML-Fab, and [125I]Fab are shown in Figure 5. Both [131I]HML-IT-Fab and [125I]HML-Fab indicated major radioactivity peaks only at the density of 1.04-1.05 mg/mL at both 10 and 30 min postinjection. On the other hand, [125I]Fab showed migration of the radioactivity peak from the fraction with the density of 1.04-1.05 mg/mL to the fraction with that of 1.10 mg/ mL from 10 to 30 min postinjection, respectively. The radioactivity peak at a density of 1.04-1.05 mg/mL was copurified with alkaline phosphodiesterase I activity, whereas that at a density of 1.10 mg/mL was copurified with β-galactosidase activity. Radiolabeled Species in the Kidney. The supernatant of each kidney homogenate was extracted with efficiencies of over 90% for all experiments. The radiolabeled species in the kidney at 10 and 30 min postinjection of radioiodinated Fab fragments were analyzed by SE-HPLC (Figure 6). Although the kidney supernatants at both 10 and 30 min after injection of [131I]HMLIT-Fab showed only two radioactivity peaks at retention times of 16-17 and 31 min, the kidney supernatants of [125I]HML-Fab showed numerous radioactivity peaks. The majority of the radioactivity in the kidney supernatant after administration of [125I]Fab was detected at a retention time similar to that of intact [125I]Fab at both postinjection time points. Figure 7 shows RP-HPLC and TLC radioactivity profiles of the kidney supernatant at 10 min after injection of [131I]HML-IT-Fab or [125I]HMLFab. The majority of the radioactivity in the kidney had a retention time of 8 min on RP-HPLC and Rf value of 0.45 on TLC, which were identical to those of miodohippuric acid (Figure 7, panels A and B). On the other hand, the kidney supernatants of [125I]HML-Fab showed

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Fujioka et al.

Figure 6. SE-HPLC radioactivity profiles of kidney homogenates at 10 and 30 min postinjection of radioiodinated Fab fragments. Under these conditions, Fab (50 kDa), cytochrome C (13 kDa), aprotinin (6 kDa), and m-iodohippuric acid (305 Da) had retention times of 16, 21, 28.5, and 31 min, respectively.

Figure 5. Subcellular fractionation of the radioactivity (b) in the kidney at 10 and 30 min after injection of radioiodinated Fab fragments. Gradients were collected in 14 fractions from the top of the gradient, assayed for the lysosomal enzyme β-galactosidase (2) and the plasma membrane enzyme alkaline phosphodiesterase I (4), and density was measured (O).

two radioactivity peaks at retention times of 6 and 8 min on RP-HPLC and Rf values of 0.1 and 0.45 on TLC, respectively (Figure 7, panels C and D). Similar radiochromatograms were obtained when the kidney supernatant obtained 30 min after injection of radioiodinated Fab fragments was analyzed (data not shown). Radiolabeled Species in the Urine. Figure 8 shows radiochromatograms of urine samples obtained for 6 h postinjection of [131I]HML-IT-Fab, [125I]HML-Fab, or [125I]Fab. On SE-HPLC analyses, the majority of the radioactivity was observed in the low molecular weight fraction with a retention time of 31 min for both [131I]HML-IT-Fab and [125I]HML-Fab (Figure 8, panels A and C). On RP-HPLC, most of the radioactivity in the urine samples was observed with a retention time (8 min) identical to that of m-iodohippuric acid for both [131I]HML-IT-Fab and [125I]HML-Fab (Figure 8, panels B and D). This was confirmed by TLC analyses of the urine samples where most of the radioactivity was observed with an Rf value identical to that of m-iodohippuric acid (data not shown). On the other hand, two radioactivity peaks were observed at retention times of 16-17 and 24 min on SE-HPLC analysis for [125I]Fab (Figure 8E). Most of the radioactivity was detected in the fraction with an Rf value (0.75) identical to that of free iodine (Figure 8F) on TLC analysis developed with solvent system a. TLC analysis using solvent system b also indicated that most

Figure 7. Radioactivity profiles of kidney homogenates at 10 min after injection of HML-conjugated Fab fragments by RPHPLC (A and C) and TLC (B and D). Samples were analyzed after filtration through a 10 kDa cutoff membrane. Under these conditions, m-iodohippuric acid had a retention time and Rf value of 8 min and 0.45, respectively.

of the radioactivity was present in the fraction with an Rf value identical to that of free iodine (data not shown). The radiochromatograms of urine samples obtained for 24 h after injection of radioiodinated Fab fragments were similar to those obtained for 6 h after injection (data not shown). For 24 h after injection of radioiodinated Fab fragments, only 2-3% of the injected radioactivity was excreted in the feces, whereas the majority of the radioactivity excreted from the body was recovered in the urine (60-70% of the injected radioactivity). Radiolabeled Species in the Plasma. Figure 9 shows SE-HPLC radiochromatograms of plasma at 10 and 30 min after injection of radioiodinated Fab fragments. Both HML-conjugated Fab fragments showed a new radioactivity peak in the high molecular weight fraction along with intact Fab fragments. The radiochromatogram of [125I]Fab showed a small increase in the radioactivity at higher and lower molecular weight fractions at 30 min.

Renal Metabolism of HML-Conjugated Fab Fragments

Figure 8. Radioactivity profiles of urine collected for 6 h after injection of radioiodinated Fab fragments by SE-HPLC (A, C, and E), RP-HPLC (B and D), and TLC (F). Urine samples were analyzed after filtration through a polycarbonate membrane with a pore diameter of 0.45 µm (A, C, and E) and after filtration through a 10 kDa cutoff membrane (B, D, and F). Under these conditions, 125I- had a retention time and Rf value of 23 min and 0.75, respectively.

Figure 9. SE-HPLC radioactivity profiles of mouse plasma at 10 and 30 min after injection of radioiodinated Fab fragments. DISCUSSION

In this study, renal metabolism of HML-conjugated Fab fragments prepared by different thiolation chemistries was compared to understand the mechanisms responsible for the low renal radioactivity levels of HMLconjugated Fab fragments. Our previous study showed

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that the spacer structure between the parental antibody molecule and the substrate (ester bonds) plays an important role in the esterase-mediated hydrolysis of ester bonds in radioimmunoconjugates (20). HML-conjugated glycoproteins prepared by different thiolation chemistries were also shown to liberate m-iodohippuric acid as the sole radiometabolite at similar rates after lysosomal proteolysis in hepatic parenchymal cells (21). On the basis of these findings, if both [131I]HML-IT-Fab and [125I]HML-Fab were metabolized primarily in the lysosomes of the renal cells, both radioiodinated Fab fragments would liberate m-iodohippuric acid as the sole radiometabolite at similar rates. If either [131I]HML-IT-Fab or [125I]HML-Fab liberates m-iodohippuric acid on the membrane fractions of renal cells by the action of brush border enzymes, different renal radioactivity levels and radiometabolites would be observed between the two HML-conjugated Fab fragments in the kidneys after administration. [125I]Fab fragments produced by direct radioiodination were used as controls to estimate lysosomal proteolysis of the antibody molecules since radiometabolites from radioiodinated proteins are rapidly eliminated from lysosomes (31-33). In biodistribution studies, [125I]Fab reached its maximum renal radioactivity levels at 30 min postinjection (Figure 4). The radioactivity in the renal cells migrated from the membrane to the lysosomal fraction from 10 to 30 min after injection of [125I]Fab (Figure 5). In addition, SE-HPLC analyses of the kidney homogenates after injection of [125I]Fab showed that while most of the radioactivity was detected as intact [125I]Fab at 10 min postinjection, small amounts of lower molecular weight radiometabolites were observed at 30 min postinjection (Figure 6). These findings suggested that Fab fragments would be transported and metabolized in the lysosomes of murine renal cells 30 min after injection into mice. Both [125I]HML-Fab and [131I]HML-IT-Fab showed significantly lower renal radioactivity levels than those of [125I]Fab as early as 10 min postinjection, as shown in Figure 4. The majority of the renal radioactivity was observed in the membrane fractions at both 10 and 30 min after injection of the two HML-conjugated Fab fragments (Figure 5). HPLC analyses of the kidney homogenates at 10 min postinjection indicated generation of m-iodohippuric acid as the major radiometabolite following administration of the two HML-conjugated Fab fragments (Figures 6 and 7). The absence of m-iodohippuric acid in the blood at 10 min after injection of both [125I]HML-Fab and [131I]HML-IT-Fab (Figure 9) indicated that the radiometabolite would be generated not in the blood but in the kidneys, as shown by our previous study (15). These findings supported our previous hypothesis that HML-conjugated Fab fragments released m-iodohippuric acid at the membrane before being reabsorbed and transported to the lysosomal compartment of the renal cells (Figure 2B). However, renal analyses demonstrated generation of different radiometabolites between the two HML-conjugated Fab fragments in the kidneys. While [131I]HMLIT-Fab registered both intact Fab and m-[131I]iodohippuric acid at 10 min postinjection, generation of additional radiometabolites was observed with [125I]HML-Fab at the same postinjection time point (Figure 7, panels C and D). Since m-iodohippuric acid was detected as the major radiometabolite in the urine after injection of both [125I]HML-Fab and [131I]HML-IT-Fab (Figure 8), the unidentified radiometabolites derived from [125I]HML-Fab would include intermediate species containing the HML moiety.

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The majority of the radioactivity was present in the membrane fraction of the renal cells at this postinjection time point of the two HML-conjugated Fab fragments (Figure 5). Thus, while [131I]HML-IT-Fab would liberate m-iodohippuric acid selectively, a few sequential reactions would be required to release the final radiometabolite, m-iodohippuric acid, from [125I]HML-Fab, which was reflected in the small but significant differences in renal radioactivity levels between [131I]HML-IT-Fab and [125I]HML-Fab at early postinjection time points (Figure 4). The glycyl-lysine sequence in [125I]HML-Fab probably hindered enzyme access to a greater extent compared with that in [131I]HML-IT-Fab due to steric interference of the parental Fab fragments as also reported previously using a radioiodination reagent with an ester bond to release m-iodohippuric acid from polypeptides (13, 20). Generation of the intermediate radiometabolites from [125I]HML-Fab suggested that brush border enzymes may have cleaved some amino acid sequences that were exposed by reducing and alkylating the disulfide bonds in the Fab fragments. These findings also indicated that it is necessary to consider the potential differences in metabolism between the two commonly used thiolation chemistries when conjugating radiolabels or other chemicals to antibodies. In conclusion, the findings of this study provided further evidence that the low renal radioactivity levels of HML-conjugated Fab from early postinjection times are attributable to rapid release of m-iodohippuric acid by the action of brush border enzymes before the fragments are incorporated into renal cells. The findings of this study also reinforced the suggestion that chemical design of radiolabeling reagents that liberate radiometabolites showing urinary excretion by the action of brush border enzymes would constitute an attractive strategy to reduce renal radioactivity levels from early postinjection times. This study suggested that chemical design of radiolabeling agents that liberate radiometabolites that are excreted into the urine from renal tubules by renal brush border enzymes but are retained in the lysosomes by lysosomal proteolysis would be useful to achieve long-term and selective target radioactivity levels using antibody fragments or peptides as vehicles regardless of whether the parental molecules are internalized into target cells. ACKNOWLEDGMENT

We are grateful to Dr. Isamu Yomoda of Diichi Radioisotope Laboratories (Tokyo) for providing Na[131I]I. This study was supported in part by Grants-in-Aid for Developing Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan. LITERATURE CITED (1) Yokota, T., Milenic, D. E., Whitlow, M., Wood, J. F., Hubert, S. L., and Schlom, J. (1993) Microautoradiographic Analysis of the Normal Organ Distribution of Radioiodinated SingleChain Fv and Other Immunoglobulin Forms. Cancer Res. 53, 3776-3783. (2) Baum, R. P., Niesen, A., Hertel, A., Adams, S., Kojouharoff, G., Goldenberg, D. M., and Hor, G. (1994) Initial Clinical Results with Technetium-99m-Labeled Ll2 Monoclonal Antibody Fragment in the Radioimmunodetection of B-Cell Lymphomas. Cancer 73, 896-899. (3) Behr, T. M., Becker, W. S., Bair, H. J., Klein, M. W., Stuhler, C. M., Cidlinsky, K. P., Wittekind, C. W., Scheele, J. R., and Wolf, F. G. (1995) Comparison of complete versus fragmented technetium-99m-labeled anti-CEA monoclonal antibodies for immunoscintigraphy in colorectal cancer. J. Nucl. Med. 36, 430-441.

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