Biodistribution of the Chimeric Monoclonal ... - ACS Publications

Division of Otolaryngology and Head & Neck Surgery, Department of Surgical Sciences, Uppsala University, Uppsala, Sweden, Division of Biomedical Radia...
0 downloads 0 Views 107KB Size
Bioconjugate Chem. 2003, 14, 805−810

805

Biodistribution of the Chimeric Monoclonal Antibody U36 Radioiodinated with a closo-Dodecaborate-Containing Linker. Comparison with Other Radioiodination Methods Marika Nestor,*,† Mikael Persson,‡ Junping Cheng,† Vladimir Tolmachev,‡ Guus van Dongen,| Matti Anniko,† and Kalevi Kairemo§ Division of Otolaryngology and Head & Neck Surgery, Department of Surgical Sciences, Uppsala University, Uppsala, Sweden, Division of Biomedical Radiation Sciences, and Division of Experimental Nuclear Medicine, Department of Oncology, Radiology, and Clinical Immunology, Uppsala University, Uppsala, Sweden, and Department of Otolaryngology/Head and Neck Surgery, Vrije Universiteit Medical Center, Amsterdam, The Netherlands. Received January 7, 2003; Revised Manuscript Received March 21, 2003

We have evaluated the applicability of the [(4-isothiocyanatobenzylammonio)undecahydro-closododecaborate (1-)] (DABI) linker molecule for antibody radiohalogenation and compared it to radiohalogenation using the linker N-succinimidyl 4-iodobenzoate (PIB) and to direct radiohalogenation using Chloramine T. These studies were performed to assess the potential of DABI conjugates and to optimize the biological properties of halogen-labeled cMAb U36. The three conjugates were evaluated in vitro for their specificity and affinity and in vivo for their biodistribution patterns in normal mice at 1.5, 6, 24, and 96 h pi. Labeling efficiencies of direct CAT labeling, indirect PIB labeling, and indirect DABI labeling were 90-95%, 60%, and 68%, respectively. This resulted in a PIB:cMAb U36 molar ratio of 1.8-2.5 and a DABI:cMAb U36 molar ratio of 4.1. The in vitro data demonstrated specific binding for all conjugates and similar affinities with values around 1 × 108 M-1. However, the in vivo data revealed accumulation of the radioiodine uptake in thyroid for the directly labeled conjugate, with a value 10 times higher than the indirectly labeled conjugates 96 h pi. Both the 125I-PIB-cMAb U36 and 125I-DABI-cMAb U36 conjugates yielded a low thyroid uptake with no accumulation, indicating different catabolites for these conjugates. This may favor the use of the indirectly labeled conjugates for future studies. Apart from the specific results obtained, these findings also demonstrate how the right linker molecule will provide additional opportunities to further improve the properties of an antibody-radionuclide conjugate.

INTRODUCTION

Radioimmunotherapy (RIT) and radioimmunodiagnosis (RID) using monoclonal antibodies (MAbs) carrying cytotoxic substances (toxin, drug, or radionuclide) is a promising and realistic approach toward improving treatment and diagnosis of cancer (1). By using radioactive nuclides as the cytotoxic substance, the problem of multidrug resistance can be avoided. Also, the crossfire effect reduces the need for every cancer cell to express the antigen, since the radiation emitted by some radionuclides can kill neighboring cancer cells. Radiohalogens are attractive candidates as radionuclides in RIT and RID since they share many chemical properties but possess a variety of half-lives and decaymodes (2). The same targeting molecule can be labeled by the same or a similar method, with a different halogen, * Correspondence should be addressed to this author at Biomedical Radiation Sciences, The Rudbeck Laboratory, S-75185 Uppsala, Sweden. E-mail: [email protected]; telephone +46 18 4713868; fax +46 18 4713432. † Division of Otolaryngology and Head & Neck Surgery, Uppsala University. ‡ Division of Biomedical Radiation Sciences, Uppsala University. § Division of Experimental Nuclear Medicine, Uppsala University. | Department of Otolaryngology/Head and Neck Surgery, Vrije Universiteit Medical Center.

depending on the biomedical problem to be solved. A γ-emitting halogen can be used for initial detection of a tumor, whereas quantification of pharmacokinetics and dosimetry can be performed using a positron-emitter such as 124I. β- or R-emitting halogens such as 131I and 211At can be applied in therapy. A tumor-seeking protein or peptide that binds to a cellular structure will most likely be internalized, either rapidly through a clathrin-dependent pathway (3) or at a slower rate through clathrin-independent endocytosis (3, 4). After internalization, the antibody-antigen complex is degraded in the lysosome through enzymatic proteolysis. If the radiocatabolite is lipophilic, it will quickly diffuse through the lipid membranes out of the cell, causing “halogen leakage” (5-7). If the labeled degradation product instead is a bulky hydrophilic or ionic compound, it cannot penetrate the cellular membrane and remains inside the cell for a longer time (2, 5-7). Consequently, attaching the nuclide to the antibody via an appropriate linker molecule will provide opportunities to optimize the in vivo properties of the targeting agent toward a high tumor uptake and retention of the label and a rapid whole-body clearance of labeled catabolites. In this aspect, indirect labeling methods through linker molecules that use hydrophilic anchor molecules may provide an advantage in comparison with direct labeling with tyrosine (using Chloramine T (CAT) or Iodogen)-labeled compounds. Also, the com-

10.1021/bc034003n CCC: $25.00 © 2003 American Chemical Society Published on Web 05/23/2003

806 Bioconjugate Chem., Vol. 14, No. 4, 2003

plementarity-determining regions (CDRs) of antibodies are at risk of being damaged through direct (CAT or Iodogen) labeling, since CDRs often are tyrosine-rich regions (8). Indirect labeling is usually performed under milder conditions but often generates lower yields and is generally a more time-consuming and complicated process. Differences between directly and indirectly radioiodinated MAbs have been observed in a number of biodistribution studies, where direct radioiodination (using CAT) have demonstrated higher accumulation of radioactivity in thyroid and stomach than indirect labeling using derivatives of benzoic acid (9-14). Studies have also shown that indirect radiohalogenation of MAbs using different linker molecules leads to differences in tissue distribution, and analysis of urine has revealed different catabolic products depending on the linker molecule used (9-14). Recently, N-succinimidyl 4-(guanidinomethyl)-3-iodobenzoate has been proposed for use in labeling antibodies with radioiodine and astatine. Studies demonstrated a 3- to 4-fold improvement of radioactivity retention in comparison to the Iodogen and 125I-SIPC (N-succinimidyl 5-iodopyridine-3-carboxylate)-labeling methods (15). However, this linker has a positive charge, which might be a disadvantage since it has been demonstrated that positively charged molecules are taken up preferentially in the kidneys, (2, 16, 17), increasing the dose burden for this radiosensitive organ. As an alternative, we propose to use polyhedral boron clusters (PBCs) as the prosthetic group. This could solve not only the problem of improving cellular retention but also that of radiocatabolite excretion. Among PBCs, boron-containing compounds such as closo-dodecaborate(2-), B12H122-, seem to be suitable for radiolabeling with halogens (18, 19). Peptides and proteins can be conjugated with various closo-dodecaborate(2-) containing ligands (20-22) showing that closo-dodecaborate can be radioiodinated in a high yield (ca. 90%) using either CAT or Iodogen. Recently, the radioiodination chemistry of B12H122- in aqueous solutions using CAT has been optimized (18), generating a rapid (>90% yield in 30 s) as well as efficient reaction in a wide range of pH (47.4). As little as 1 nmol of closo-dodecaborate can be iodinated with a high yield, forming highly stable boronhalogen bonds. These aspects, together with the negative charge on the dodecaborate cluster and the rapid excretion via the kidneys (19), make the B12H122- compounds promising as pendant groups for radiohalogenation of tumor-targeting substances for radionuclide diagnostics and therapy of cancer. DABI, a derivative of closo-dodecaborate, [(4-isothiocyanatobenzylammonio)undecahydro-closo-dodecaborate (1-)], has recently been successfully radioiodinated at our laboratory with a high yield (93-95%) and coupled to a herceptin antibody (23, 24). This prompted us to further investigate this compound as a pendant group for radiohalogen labeling of antibodies and to compare its biodistribution with other direct and indirect labeling methods. The chimeric monoclonal antibody (cMAb) used in these studies was cMAb U36, appearing to have much potential in RIT toward head and neck squamous cell carcinomas (HNSCCs) (25-27). The cMAb U36 recognizes the CD44 splice variant CD44v6, located on the outer cell surface (28). Clinical biodistribution studies evaluated by radioimmunoscintigraphy and by biopsy measurements in head and neck cancer patients have shown high and selective accumulation of cMAb U36 in

Nestor et al.

Figure 1. Structures of the three different 125I-antibody conjugates used. In the directly (CAT) labeled antibody (a), the 125I is directly attached to the tyrosine residues of the antibody. In the indirectly PIB-labeled conjugate (b), the 125I is connected to the antibody via the PIB linker molecule to the lysine residues of the antibody. In the case of the indirectly DABI-labeled conjugate (c), the 125I is attached via the DABI linker molecule to the lysine residues of the antibody.

primary tumors and lymph node metastases (29). Selective tumor accumulation to nude mice bearing human HNSCC xenografts has been demonstrated for the 125Ilabeled cMAb U36 (using Iodogen), as well as for 131I- and 186Re-labeled MAb U36 (30-34), and recently the chimeric MAb U36 was evaluated in two clinical RIT trials using 186Re as a therapeutic radionuclide (25, 27). In the present study, the cMAb U36 has been labeled with 125I using three different labeling techniques, i.e., direct labeling using CAT, and indirect labeling using either DABI or PIB (N-succinimidyl 4-iodobenzoate) linker molecules. The goal of the study was to investigate how indirect iodination of cMAb U36 using DABI affects the specificity, affinity, and biodistribution of the antibody compared to the same antibody labeled using CAT and PIB. These data were sought to investigate the feasibility of DABI as a linker molecule, as well as to optimize the biological properties of halogen-labeled cMAb U36 for possible future use in RIT and RID. EXPERIMENTAL PROCEDURES

Cell Lines. The HNSCC cell lines SCC-9 and SCC-25 (obtained from American Type Culture Collection) were cultured in a 1:1 mixture of Ham’s F12 and Dulbecco’s Modified Eagle Medium (DMEM), supplemented with 10% foetal calf serum, 0.4 mM hydrocortisone, 2 mM L-glutamine and antibiotics (100 IU penicillin and 100 µg/mL streptomycin). Cells were incubated at 37 °C in an atmosphere containing humidified air with 5% CO2. Cells were trypsinized and grown in separate dishes used for experiments 3-4 days prior to the studies. Antibody. The chimeric monoclonal antibody used in these studies was cMAb U36. The selection and production of the antibody has been described previously (32, 35). The antibody was first stored in citrate buffer and then separated by size-exclusion chromatography on a NAP-5 column preequilibrated with purified (ELGA) water. It was then freeze-dried overnight and stored in -20 °C. Direct Labeling. Freshly prepared solution of cMAb U36 in PBS (60 µL, 5 mg/mL) was mixed with 125I iodine solution (5 MBq). Reaction was initiated by adding CAT in PBS (10 µL, 2 mg/mL) and was quenched after rigorous vortexing during 5 min by adding sodium metabisulfite (20 µL, 2 mg/mL). Labeled antibody (125I-cMAb U36) was separated from nonreacted 125I and low-molecular-weight reaction components by size-exclusion chromatography on a NAP-5 column preequilibrated with PBS. See Figure 1a) for the structure of the directly radioiodinated antibody.

Evaluation of a closo-Dodecaborate-Containing Linker

PIB Labeling. Stock solution of 125I (45 MBq in 15 µL) was mixed with 10 µL of 0.1% acetic acid, and a solution of N-succinimidyl 4-(trimethylstannyl)benzoate in 5% acetic acid in methanol (5 µL, 1 mg/mL) prepared as described in (36) was then added. Labeling was started by adding CAT in water (10 µL, 2 mg/mL) and was quenched after 5 min of vortexing by adding sodium metabisulfite (10 µL, 4 mg/mL water). Freshly prepared solution of cMAb U36 in borate buffer, pH 9.1 (30 µL, 10 mg/mL) was added. The solution was stirred carefully, and the coupling reaction proceeded during 30 min at 37 °C. Separation of labeled cMAb U36 (125I-PIB-cMAb U36) was performed by size-exclusion chromatography on a NAP-5 column preequilibrated with PBS. See Figure 1b) for the structure of the indirectly PIB-radioiodinated antibody. DABI Labeling. A solution of potassium salt of DABI in water (3 µL, 1 mg/mL), prepared as described in (37), was mixed with stock solution of 125I (60 MBq in 20 µL). Labeling was started by adding CAT in water (10 µL, 2 mg/mL) and was quenched after 5 min of vortexing by adding sodium metabisulfite (10 µL, 4 mg/mL water). Freshly prepared solution of cMAb U36 in borate buffer, pH 9.1 (30 µL, 10 mg/mL) was then added. The solution was carefully stirred, and the coupling reaction proceeded during 30 min at 37 °C. Separation of labeled cMAb U36 (125I-DABI-cMAb U36) was performed by size-exclusion chromatography on a NAP-5 column preequilibrated with PBS. See Figure 1c) for the structure of the indirectly DABI-radioiodinated antibody. Affinity Measurements. Kinetic affinity constants were measured using saturation curves with a fixed amount of cells and different concentrations of labeled antibody. Six dishes per data point containing approximately 50 000 cells of SCC9 or SCC25 HNSCC cells were prepared. An excess of unlabeled antibody was added to three of the dishes per data point for unspecific binding correction. These dishes also functioned as specificity controls of the antibody, since the binding of a labeled antibody conjugate that has preserved its antigen specific binding will be blocked in the dishes containing an excess of unlabeled antibody. Concentrations varying between 0.01 and 7 µg of 125I labeled cMAb U36 (125I-cMAb U36, 125I-PIB-cMAb U36, or 125I-DABIcMAb U36) was then added to all dishes. Cells were incubated on ice (in order to prevent endocytosis) for 15 h. The incubation medium was then collected, and the dishes were washed six times with HAM’s F12/DMEM medium. The cells were detached with 0.5 mL of trypsinEDTA solution for 10 min in 37 °C and resuspended in 1 mL complete culture medium. Cell counting was performed on 0.5 mL of the suspension, and radioactivity measurements were performed on the remaining 1 mL in a gamma well-counter. The values were analyzed using GraphPad Prism in order to construct saturation curves and to calculate the affinity. Antibody Biodistribution. All three in vivo studies were carried out in normal mice (adult female NMRI mice, BK Universal AB, Stockholm) with appropriate licenses from The Local Ethics Committee for Animal Research. Animals were housed at the Rudbeck animal facility and were allowed to acclimatize to the facility for at least a week before the start of experiments. Groups of four mice per time point were injected intravenously via tail vein with 15 µg of 100-120 kBq 125I-labeled cMAb U36 diluted in PBS. Animals were sacrificed at 1.5, 6, 24, and 96 h after injection. At indicated time points, mice were anaesthetized, heart punctured, killed, and dissected. Urine and blood were collected, as well as the

Bioconjugate Chem., Vol. 14, No. 4, 2003 807 Table 1. cMAb U36 Affinity as a Function of Radioiodination Method in Two HNSCC Cell Lines

radioiodination technique

affinity, cell line SCC9 (M-1)a

affinity, cell line SCC25 (M-1)a

direct (CAT) labeling indirect PIBb labeling indirect DABIc labeling

0.6 ( 0.3 × 108 1.0 ( 0.2 × 108 0.8 ( 0.3 × 108

0.4 ( 0.3 × 108 1.5 ( 0.6 × 108 1.2 ( 0.1 × 108

a Mean ( SD (n ) 3). b N-Succinimidyl 4-iodobenzoate. c (4Isothiocyanatobenzylammonio)undecahydro-closo-dodecaborate (1-).

major organs and samples of brain, muscle, skeleton, and skin. The samples were then weighed, and the activity was measured. To obtain a quantitative measure of the injected radioactivity, the radioactivity of each syringe was measured before and after injection, and the radioactivity in the tails was measured after dissection. The activity expressed in percentage injected dose per gram of tissue was then calculated, and the average values were used to construct biodistribution curves. The Kruskal-Wallis test was performed in MINITAB to determine the significance of deviating values (significant if P < 0.05). RESULTS

Labeling. Direct CAT labeling of cMAb U36 was performed with a labeling efficiency of 90-95%. PIB labeling was performed with a labeling efficiency of 60%, resulting in a PIB:cMAb U36 molar ratio of 1.8-2.5. DABI labeling was performed with a labeling efficiency of 68% and resulted in a DABI:cMAb U36 molar ratio of 4.1. Affinity Measurements. The affinities of the three antibody conjugates are shown in Table 1. The differences in affinity for the three conjugates were small, with values around 1 × 108 M-1. Antigen specific binding of all three conjugates were clearly demonstrated in both HNSCC cell lines, as the binding of conjugates was prevented by an excess of unlabeled antibody (data not shown). Biodistribution. The biodistribution of 125I-cMAb U36, 125I-DABI-cMAb U36, and 125I-PIB-cMAb U36 in various organs are shown in Figures 2 and 3. Figure 2 shows the organs in which no significant biodistribution difference (P g 0.05) between the conjugates could be seen, and Figure 3 shows the organs in which a significant difference (P < 0.05) could be seen for at least one of the conjugates in at least one time point. Generally, amounts in blood and organs decreased with time, with the exception for thyroid and skin. The lowest uptake was seen in brain for all three compounds. The indirectly labeled conjugates showed minor differences in tissue distribution, except in the reticuloendothelial system (i.e., liver and spleen), where the 125I-DABI-cMAb U36 conjugate demonstrated a higher uptake in the 1.5 and 96 h time points (see Table 2). At the 96 h time point, the 125I-DABI-cMAb U36 conjugate displayed a 2.2 and 2.9 times higher uptake than the other conjugates in liver and spleen, respectively. The 125I-DABI-cMAb U36 conjugate also displayed a higher amount of radioactivity in blood than the other conjugates at the 96 h time point. However, the most pronounced difference in uptake can be seen in thyroid starting from the 24 h time point and increasing even more at the 96 h time point. With the direct labeling technique, a radioactivity uptake corresponding to 10 times the value of the indirectly labeled compounds can be observed at the 96 h time point (see Figure 3). The thyroid uptake is clearly increasing with

808 Bioconjugate Chem., Vol. 14, No. 4, 2003

Nestor et al.

Figure 3. Comparison of the biodistribution of 125I-cMAb U36 (×), 125I-DABI-cMAb U36 (0), and 125I-PIB-cMAb U36 (O) in blood, liver, spleen, and thyroid. Animals were sacrificed at 1.5, 6, 24, and 96 h after injection. The unit % ID/g refers to the activity expressed in percentage injected dose per gram of tissue. Significant differences (P < 0.05) could be observed at the 1.5 and 96 h time points in liver and spleen and the 96 h time point in blood, where the DABI conjugate displayed higher values. In thyroid, the directly labeled conjugate displayed significantly higher values than the indirectly labeled conjugates at the 24 and 96 h time points. Error bars represent standard deviations (N ) 4). Table 2. Comparison of the % ID/g Tissuea of 125I-cMAb U36, 125I-DABI-cMAb U36, and 125I-PIB-cMAb U36 in Liver and Spleen organ liver

spleen

time point direct (CAT) indirect PIBb indirect DABIc (h) labeling labeling labeling 1.5d 6 24 96d 1.5d 6 24 96d

3.6 ( 1.6 3.5 ( 1.4 2.6 ( 0.9 1.5 ( 0.9 3.1 ( 1.0 2.7 ( 0.9 1.7 ( 0.8 1.1 ( 0.8

5.2 ( 2.3 4.4 ( 1.0 2.1 ( 0.6 1.6 ( 0.6 3.2 ( 1.6 4.3 ( 1.6 1.3 ( 0.3 1.1 ( 0.4

14.1 ( 1.7 4.8 ( 4.5 4.0 ( 2.0 3.4 ( 0.2 11.9 ( 2.2 7.1 ( 4.2 4.1 ( 2.5 3.1 ( 0.8

a The unit % ID/g refers to the activity expressed in percentage injected dose per gram of tissue. Errors represent standard deviations (N ) 4). b N-Succinimidyl 4-iodobenzoate. c (4-isothiocyanatobenzylammonio)undecahydro-closo-dodecaborate (1-). d The DABI conjugate displayed significantly (P < 0.05) higher values than the other conjugates at these time points.

Figure 2. Comparison of the biodistribution of 125I-cMAb U36 (×), 125I-DABI-cMAbU36 (0), and 125I-PIB-cMAb U36 (O) in various organs. Animals were sacrificed at 1.5, 6, 24, and 96 h after injection. The unit % ID/g refers to the activity expressed in percentage injected dose per gram of tissue. In these organs, no significant difference (P g 0.05) between the conjugates could be observed. No accumulation could be seen in any organs except in skin, where some accumulation could be observed from the 24 h time point. Error bars represent standard deviations (N ) 4).

time for this conjugate, whereas the indirectly labeled conjugates show low and constant thyroid values. DISCUSSION

In this study we have conducted a comparative investigation of three radiohalogenation methods on the cMAb

U36. By studying the labeling effect on specificity, affinity, and biodistribution, the biological suitability of the cMAb U36 conjugates could be evaluated. Moreover, this paper describes the very first in vivo evaluation of DABI in order to assess its applicability for continued studies. The data obtained in vitro showed antigen specific binding and minor differences in affinity for all conjugates with values around 1 × 108 M-1. The in vivo data pointed out a 10 times higher accumulation in thyroid for the directly labeled conjugate 96 h pi. This is consistent with a number of studies where direct radioiodination (using CAT) exhibits higher accumulation of radioactivity in thyroid than indirect labeling using derivatives of benzoic acid (9-13). Since the thyroid is known to accumulate free radioiodide, this could indicate a different catabolic pathway for the directly labeled conjugate. It has also been hypothesized that the lesser uptake of radiolabel in thyroid for the indirectly labeled compounds via benzoic derivatives is due to rapid urinary excretion of the intracellular catabolic products. This quick excretion may prevent further in vivo transforma-

Evaluation of a closo-Dodecaborate-Containing Linker

tion of the catabolites and the release of free halide, resulting in a lower thyroid uptake (13, 14). The in vivo data also demonstrated a higher level of 125I-DABI-cMAb U36 in liver and spleen compared to the other conjugates. This increase might raise some concerns about its use, even though 3.4 and 3.1% ID/g tissues in liver and spleen at the last time point is not alarmingly high. However, this higher level could indicate residualization of the DABI linker, and future xenograft studies will have to assess if the amount of residualization obtained in tumors outweigh any disadvantage of increased uptake in liver and spleen. The increasing uptake in skin seen in this study for all three conjugates (see Figure 2) is intriguing, since the CD44v6 epitope recognized by cMAb U36 is a human epitope and should not be reactive with CD44v6 splice variants in mice. However, it has been shown for mice that skin is an important site for catabolism for the IgG1 subclass (38, 39), and the epithelial uptake seen in skin in our study could be caused by this. Also, the possibility that the high skin levels were caused by urine or blood contamination cannot be ruled out, although all samples were washed before measurements. In conclusion, among three differently radioiodinated cMAb U36 conjugates, all three conjugates showed high specific binding and similar affinities but displayed different biodistribution patterns. The directly labeled conjugate showed clear and high accumulation in thyroid, in contrast to the indirectly labeled conjugates. Both 125IPIB-cMAb U36 and 125I-DABI-cMAb U36 showed a consistently low thyroid uptake, indicating high stability of the radiolabels and different radiocatabolites from the directly labeled conjugate. This may favor the use of these conjugates for future studies. For studies where thyroid uptake can be neglected, the direct CAT labeling method might be acceptable with unaffected affinity, offering a fast and simple labeling method with high yield. However, when the tumor target is in the throat area, the uptake in thyroid will complicate diagnosis if the tumor uptake cannot be distinguished from the thyroid uptake, and dose planning for RIT may be impossible. If instead the uptake in thyroid is low, medication to block radionuclide uptake can be avoided and the RIT process simplified. Also, the risk of causing hypothyroidism in the patient can be reduced. Since the cMAb U36 previously has been shown to specifically target HNSCC xenograft models, the fact that the linker molecules did not considerably alter the specific binding or the affinity of the antibody is promising. This study also indicates that radioiodinated DABI conjugated to an appropriate tumor-seeking molecule can provide a stable residualizing iodine label which, if applied to a relevant targeting agent, will be of interest for imaging and therapy. Apart from the specific results obtained, these findings also demonstrate how the proper linker molecule will provide additional opportunities to further improve the properties of an antibody-radionuclide conjugate. ACKNOWLEDGMENT

The authors wish to thank Dr. Igor Sivaev who prepared the potassium salt of DABI, and Dr. Anna Orlova who prepared N-succinimidyl 4-(trimethylstannyl)benzoate. We also wish to thank Dr. Lars Gedda for supervising the affinity experiments and Qichun Wei for help with the data collection in the DABI biodistribution experiment. This work was partly supported by grants

Bioconjugate Chem., Vol. 14, No. 4, 2003 809

from Cancerfonden, Sweden (Project numbers 4462-B0102PAA and 3980-B00-04XBB). LITERATURE CITED (1) Potamianos, S., Varvarigou, A. D., and Archimandritis, S. C. (2000) Radioimmunoscintigraphy and radioimmunotherapy in cancer: principles and application. Anticancer Res. 20, 925-948. (2) Tolmachev, V., and Sjo¨berg, S. (2002) Polyhedral boron compounds as potential linkers for attachment of radiohalogens to targeting proteins and peptides. A review. Collect Czech. Chem. Commun. 67, 913-935. (3) van Deurs, B., Petersen, O. W., Olsnes, S., and Sandvig, K. (1989) The ways of endocytosis. Int. Rev. Cytol. 117, 131177. (4) Kyriakos, R. J., Shih, L. B., Ong, G. L., Patel, K., Goldenberg, D. M., and Mattes, M. J. (1992) The fate of antibodies bound to the surface of tumor cells in vitro. Cancer Res. 52, 835-842. (5) Geissler, F., Anderson, S. K., Venkatesan, P., and Press, O. (1992) Intracellular catabolism of radiolabeled anti-mu antibodies by malignant B-cells. Cancer Res. 52, 2907-2915. (6) Press, O. W., Shan, D., Howell-Clark, J., Eary, J., Appelbaum, F. R., Matthews, D., King, D. J., Haines, A. M., Hamann, P., Hinman, L., Shochat, D., and Bernstein, I. D. (1996) Comparative metabolism and retention of iodine-125, yttrium-90, and indium-111 radioimmunoconjugates by cancer cells. Cancer Res. 56, 2123-2129. (7) Stein, R., Goldenberg, D. M., Thorpe, S. R., and Mattes, M. J. (1997) Advantage of a residualizing iodine radiolabel for radioimmunotherapy of xenografts of human nonsmall-cell carcinoma of the lung. J. Nucl. Med. 38, 391-395. (8) Nikula, T. K., Bocchia, M., Curcio, M. J., Sgouros, G., Ma, Y., Finn, R. D., and Scheinberg, D. A. (1995) Impact of the high tyrosine fraction in complementarity determining regions: measured and predicted effects of radioiodination on IgG immunoreactivity. Mol. Immunol. 32, 865-872. (9) Wilbur, D. S., Hadley, S. W., Hylarides, M. D., Abrams, P. G., Beaumier, P. A., Morgan, A. C., Reno, J. M., and Fritzberg, A. R. (1989) Development of a stable radioiodinating reagent to label monoclonal antibodies for radiotherapy of cancer. J. Nucl. Med. 30, 216-226. (10) Zalutsky, M. R., and Narula, A. S. (1987) A method for the radiohalogenation of proteins resulting in decreased thyroid uptake of radioiodine. Int. J. Radiat. Appl. Instrum. [A] 38, 1051-1055. (11) Zalutsky, M. R., and Narula, A. S. (1988) Radiohalogenation of a monoclonal antibody using an N-succinimidyl 3-(trin-butylstannyl)benzoate intermediate. Cancer Res. 48, 14461450. (12) Zalutsky, M. R., Noska, M. A., Colapinto, E. V., Garg, P. K., and Bigner, D. D. (1989) Enhanced tumor localization and in vivo stability of a monoclonal antibody radioiodinated using N-succinimidyl 3-(tri-n-butylstannyl)benzoate. Cancer Res. 49, 5543-5549. (13) Hoglund, J. (2002) On the use of 76Br-labelled Monoclonal Antibodies for PET. Preclinical Evaluation of Halogenated Antibodies for Diagnosis and Treatment of Cancer. Acta Univ. Ups., Uppsala Diss. Fac. Med. (14) 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, 111-116. (15) Vaidyanathan, G., Affleck, D. J., Li, J., Welsh, P., and Zalutsky, M. R. (2001) A polar substituent-containing acylation agent for the radioiodination of internalizing monoclonal antibodies: N-succinimidyl 4-guanidinomethyl-3-[131I]iodobenzoate ([131I]SGMIB). Bioconjugate Chem. 12, 428-438. (16) Behr, T. M., Becker, W. S., Sharkey, R. M., Juweid, M. E., Dunn, R. M., Bair, H. J., Wolf, F. G., and Goldenberg, D. M. (1996) Reduction of renal uptake of monoclonal antibody fragments by amino acid infusion. J. Nucl. Med. 37, 829833. (17) Behr, T. M., Sharkey, R. M., Juweid, M. E., Blumenthal, R. D., Dunn, R. M., Griffiths, G. L., Bair, H. J., Wolf, F. G.,

810 Bioconjugate Chem., Vol. 14, No. 4, 2003 Becker, W. S., and Goldenberg, D. M. (1995) Reduction of the renal uptake of radiolabeled monoclonal antibody fragments by cationic amino acids and their derivatives. Cancer Res. 55, 3825-3834. (18) Tolmachev, V., Lundqvist, H., Carlsson, J., Sivaev, I., Orlova, A., and Sundin, A. (1997) Labeling and in vivo evaluation of closo-dodecaborate as a linker for attachment of iodine to radiopharmaceuticals. J. Labelled Compd. Radiopharm. 40, 125. (19) Orlova, A., Sivaev, I., Sjoberg, S., and Tolmachev, V. (2001) Radioiodination of monocarboranes. 2nd Eur. Symp. Boron Chem. (EUROBORON 2), (Book of Abstracts) 33. (20) Tolmachev, V., Koziorowski, J., Sivaev, I., Lundqvist, H., Carlsson, J., Orlova, A., Gedda, L., Olsson, P., Sjoberg, S., and Sundin, A. (1999) Closo-dodecaborate(2-) as a linker for iodination of macromolecules. Aspects on conjugation chemistry and biodistribution. Bioconjugate Chem. 10, 338-345. (21) Orlova, A., Tolmachev, V., and Lundqvist, H. (2000) Closododecaborate (2-) anion as a potential prosthetic group for attachment of radioiodine to proteins. Aspects of labeling chemistry in aqueous solutions. Eur. J. Nucl. Med. 27, 1210. (22) Tolmachev, V., Bruskin, A., Sivaev, I., Lundqvist, H., and Sjoberg, S. (2001) Oxidative radiobromination of the dodecahydro-closo-dodecaborate (2-) anion. 2nd Eur. Symp. Boron Chem. (EUROBORON 2), (Book of Abstracts) 55. (23) Tolmachev, V., Bruskin, A., Winberg, K. J., Sivaev, I., Persson, M., Lundqvist, H., Sjoberg, S., and Carlsson, J. (2002) The use of derivatives of polyhedral boron anions (PHA), closo-dodecaborate and nido-carborate, for radiobromination of anti-HER-2 antibody Herceptin for immunoPET. (Abstracts of the 15th meeting of the International Research Group in Immunoscintigraphy and Immunotherapy (IRIST), Rotterdam, The Netherlands, May 24-25 May, 2002) Cancer Biother. Radiopharm. 17, 353-354. (24) Tolmachev, V., Orlova, A., Bruskin, A., Sivaev, I., Persson, M., Sjoberg, S., Carlsson, J., and Lundqvist, H. (2002) The use of benzyl isothiocyanate derivative of closo-dodecaborate dianion for indirect radioiodination and radiobromination of monoclonal antibodies. Eur. J. Nucl. Med. 29, Supplement 1: S76. (25) Colnot, D. R., Ossenkoppele, J. C., Quak, J. J., de Bree, R., Bo¨rjesson, P. C., Snow, G. B., and van Dongen, G. A. (2002) Re-infusion of Unprocessed, G.-CSF-Stimulated Whole Blood Allows Dose Escalation of 186-Re-cMAb U36 Radioimmunotherapy in a Phase I Dose Escalation Study. Clin. Cancer Res. 8, 3401-3406. (26) Colnot, D. R., Quak, J. J., Roos, J. C., de Bree, R., Wilhelm, A. J., Snow, G. B., and van Dongen, G. A. (2001) Radioimmunotherapy in patients with head and neck squamous cell carcinoma: initial experience. Head Neck 23, 559-565. (27) Colnot, D. R., Quak, J. J., Roos, J. C., van Lingen, A., Wilhelm, A. J., van Kamp, G. J., Huijgens, P. C., Snow, G. B., and van Dongen, G. A. (2000) Phase I therapy study of 186Re-labeled chimeric monoclonal antibody U36 in patients with squamous cell carcinoma of the head and neck. J. Nucl. Med. 41, 1999-2010. (28) Van Hal, N. L., Van Dongen, G. A., Rood-Knippels, E. M., Van Der Valk, P., Snow, G. B., and Brakenhoff, R. H. (1996)

Nestor et al. Monoclonal antibody U36, a suitable candidate for clinical immunotherapy of squamous-cell carcinoma, recognizes a CD44 isoform. Int. J. Cancer 68, 520-527. (29) de Bree, R., Roos, J. C., Quak, J. J., den Hollander, W., Snow, G. B., and van Dongen, G. A. (1995) Radioimmunoscintigraphy and biodistribution of technetium-99m-labeled monoclonal antibody U36 in patients with head and neck cancer. Clin. Cancer Res. 1, 591-598. (30) Vrouenraets, M. B., Visser, G. W. M., Stigter, M., Oppelaar, H., Snow, G. B., and van Dongen, G. A. M. S. (2001) Targeting of Aluminum (III) Phthalocyanine Tetrasulfonate by Use of Internalizing Monoclonal Antibodies: Improved Efficacy in Photodynamic Therapy. Cancer Res. 61, 1970-1975. (31) Vrouenraets, M. B., Visser, G. W., Loup, C., Meunier, B., Stigter, M., Oppelaar, H., Stewart, F. A., Snow, G. B., and van Dongen, G. A. (2000) Targeting of a hydrophilic photosensitizer by use of internalizing monoclonal antibodies: A new possibility for use in photodynamic therapy. Int. J. Cancer 88, 108-114. (32) Schrijvers, A. H., Quak, J. J., Uyterlinde, A. M., van Walsum, M., Meijer, C. J., Snow, G. B., and van Dongen, G. A. (1993) MAb U36, a novel monoclonal antibody successful in immunotargeting of squamous cell carcinoma of the head and neck. Cancer Res. 53, 4383-4390. (33) van Gog, F. B., Brakenhoff, R. H., Stigter-van Walsum, M., Snow, G. B., and van Dongen, G. A. (1998) Perspectives of combined radioimmunotherapy and anti-EGFR antibody therapy for the treatment of residual head and neck cancer. Int. J. Cancer 77, 13-18. (34) van Gog, F. B., Visser, G. W., Stroomer, J. W., Roos, J. C., Snow, G. B., and van Dongen, G. A. (1997) High dose rhenium-186-labeling of monoclonal antibodies for clinical application: pitfalls and solutions. Cancer 80, 2360-2370. (35) Brakenhoff, R. H., van Gog, F. B., Looney, J. E., van Walsum, M., Snow, G. B., and van Dongen, G. A. (1995) Construction and characterization of the chimeric monoclonal antibody E48 for therapy of head and neck cancer. Cancer Immunol. Immunother. 40, 191-200. (36) Koziorowski, J., Henssen, C., and Weinreich, R. (1998) A new convenient route to radioiodinated N-succinimidyl 3- and 4-iodobenzoate, two reagents for radioiodination of proteins. Appl. Radiat. Isot. 49, 955-959. (37) Sivaev, I., Bruskin, A., Nesterov, V. V., Antipin, M. Y., Bregadze, V. I., and Sjoberg, S. (1999) Synthesis of Schiff Bases Derived from the Ammoniaundecahydro-closo-dodecaborate(1-) Anion, [B12H11NHdCHR]-, and Their Reduction into Monosubstituted Amines [B12H11NH2CH2R]-: A New Route to Water Soluble Agents for BNCT. Inorg. Chem. 38, 5887-5893. (38) Henderson, L. A., Baynes, J. W., and Thorpe, S. R. (1982) Identification of the sites of IgG catabolism in the rat. Arch. Biochem. Biophys. 215, 1-11. (39) Moldoveanu, Z., Epps, J. M., Thorpe, S. R., and Mestecky, J. (1988) The sites of catabolism of murine monomeric IgA. J. Immunol. 141, 208-213.

BC034003N