ORIGINAL ARTICLE 
Annals of Nuclear Medicine Vol. 14, No. 3, 173-179, 2000 
Rhenium-188-labeled anti-neural cell adhesion molecule antibodies with 2-iminothiolane modification for targeting small-cell lung cancer 
Masako N. HOSONO,* Makoto HOSONO,** Anil K. MISHRA,*** Alain FAIVRE-CHAUVET,*** Emmanuel GAUTHEROT,**** Jacques BARBET,**** F.F. (Russ) KNAPP, Jr.***** and Jean-Francois CHATAL*** 
*Department of Radiology, Osaka City University **Department of Radiology, Saitama Medical Center ***INSERM Research Unit 463, Nantes, France ****Immunotech SA, Marseille, France *****Nuclear Medicine Program, Oak Ridge National Laboratory, Oak Ridge, TN, USA 
We have evaluated the potential of 188Re-labeled monoclonal antibodies (MAbs) modified with 2-iminothiolane (2IT) for targeting small-cell lung cancer (SCLC). Radiolabeled MAbs NK1NBL1 and C218 recognizing neural cell adhesion molecule were injected i.v. into athymic mice inoculated with human SCLC tumors, and the biodistribution was examined. NK1NBL1 localized in the tumors better than C218. 188Re-labeled MAbs cleared from the blood faster than 125I-labeled counterparts, resulting in higher tumor-to-blood ratios. In conclusion, the 188Re-labeled MAbs are attractive candidates for imaging and therapy of SCLC. 
Key words: rhenium-188, monoclonal antibody, small-cell lung cancer, neural cell adhesion molecule, 2-iminothiolane 
INTRODUCTION 
SMALL-CELL LUNG CANCER (SCLC) is characterized by endocrine features, a tendency to metastasize, high but temporary chemo- and radiosensitivity, and poor prognosis. The mortality of patients with SCLC remains more than 90% at 2 years after diagnosis.1 As most post-therapy patients suffer from recurrence, and tumors become refractory to repeated therapy, antibody-guided internal radiotherapy has been expected as a further therapeutic strategy. Several reports have described animal studies of SCLC therapy with radiolabeled monoclonal antibodies (MAbs) reactive with some SCLC-related antigens.2,3 Neural cell adhesion molecule (NCAM) is considered as the most specific among the SCLC-related antigens. Since most SCLC tumors express NCAM on the surface membrane of the cells,4 it is thought to be an optimal target for 
Received January 12, 2000, revision accepted March 1, 2000. For reprint contact: Makoto Hosono, M.D., Ph.D., Department of Radiology, Saitama Medical Center, 1981 Kamoda, Kawagoe, Saitama 350-8550, JAPAN. E-mail: makohoso@saitama-med.ac.jp 
localizing SCLC tumors.
 In terms of cost effectiveness for imaging and internal radiation therapy, 188Re is a promising radionuclide. It has 155 keV gamma-emission suitable for scintigraphy and the maximum b-energy of 2.1 MeV effective for radioimmunotherapy with a half-life of 16.98 h. Rhenium-188 is also readily available from the 188W/188Re generator as carrier-free perrhenate (the half-life of 188W is 69.4 d). Rhenium-188 has good clinical potential for radiation synovectomy,5 pain relief in patients with multiple bone metastases as 188Re-hydroxyethyliden diphosphonate (HEDP),6 and inhibition of restenosis after percutaneous transcatheter coronary angioplasty.7
 To date, methods for radiolabeling antibodies with radioisotopes of rhenium, both directly and indirectly, have been proposed. Direct methods, where the endogenous complexing groups are used, may be susceptible to low label stability.8-10 Indirect methods with chelating agents11,12 may improve stability of rhenium-labeled antibodies, but direct methods have the advantage of simple preparation, which is suitable for administration to patients. 
 The combination of 188Re as a radiolabel, anti-NCAM MAbs, and a direct labeling technique, could therefore be a potent tool for the diagnosis and therapy of SCLC. Although we adapted a direct labeling technique in this study, we modified antibodies with 2-iminothiolane (2IT) which provides a thiol reactive position for the attachment of rhenium in order to obtain stable labeling. This technique has already proved effective in achieving stable labeling of antibodies with 99mTc.13-15 In this study we evaluated the effectiveness of 2IT modification for 188Re-labeling of anti-NCAM MAbs in vitro and in a mouse model bearing human SCLC. 
MATERIALS AND METHODS 
Antibodies 
The murine MAbs C21816 and NK1NBL117 (IgG1s), reacting with human NCAM, were supplied by Immunotech (Marseille, France). The MAb OC125, used as a nonspecific Ab in this study, was generated by immunizing mice with human ovarian serous cystadenocarcinoma and recognized a high-molecular weight glycoprotein CA125.18 
Cell line and xenografts 
The SCLC cell line NCI-H69 cells,19 obtained from the American Type Culture Collection (Manassas, VA, USA), were cultured in RPMI 1640 culture medium (Life Technologies, Grand Island, NY, USA) supplemented with 1 mM glutamine and 10% fetal calf serum. For the studies in mice, NCI-H69 SCLC cells were implanted by s.c. inoculation of a tumor mince into the flanks of 5 to 7 week-old female BALB/c nu/nu mice. Xenografted mice were used when the tumor volume reached about 0.5 cm3, 2-3 weeks after inoculation. 
Radiolabeling of antibodies 
Rhenium-188 perrhenate solution was obtained from a 188W/188Re generator available from the Oak Ridge National Laboratory (Oak Ridge, TN, USA).20 Before labeling with 188Re, MAbs were derivatized by 2IT (molecular weight of 101, Fig. 1 ), which provides thiol-reactive sites for the attachment of rhenium without splitting the disulfide bonds of the antibody molecules. Briefly, to modify MAbs, 500 ug (3.3 nmol) of intact MAbs C218, NK1NBL1, and OC125 mixed with 50 ul of 2IT solution (Sigma Chemical Co., St. Louis, MO, USA, 2.5 mg i.e. 25 umol of 2IT in 50 ul of dimethylsulfoxide), and 0.1 M phosphate buffer (PB) pH 7 (totally 1 ml) were incubated for 15 min at room temperature. For reduction of 188Re, 1.35 ml of eluted 188Re perrhenate with a specific volume of 185 MBq/ml and 7.5 ml of 1 mM SnCl2 and 0.15 M Na4P2O7 solution were incubated for 1 h at 100deg.C. Then, to label MAbs with 188Re, reduced 188Re and pretreated MAbs were incubated for 16 h at 4deg.C. The 188Re-labeled MAbs were purified by Sephadex G-25 gel chromatography (Pharmacia LKB Biotechnology, Uppsala, Sweden). Labeling efficiency was 36.4-57.8% for the 188Re-labeled MAbs. The specific activity for 188Re-labeled C218, NK1NBL1, and OC125 was 97-126 MBq/mg. 
 The intact MAbs C218, NK1NBL1, and OC125 were labeled with 125I by the chloramine-T method. MAbs (40 ug) in 0.3 M PB pH 7.5, and 125I (11.1 MBq) for protein labeling were mixed with 2.5 ug of chloramine-T (Aldrich, St. Quentin Fallvier. France) dissolved in 0.3 M PB. 5 min later the radiolabeled MAb was separated from free radioiodine by Sephadex G-25 gel chromatography. The specific activity of 125I-labeled C218, NK1NBL1, and OC125 was 210-282 MBq/mg. 
Stability 
To verify that the 188Re-labeled C218 and NK1NBL1 were not fragmented during the reduction-mediated radiolabeling, 188Re-labeled C218 and NK1NBL1 were analyzed by size exclusion high performance liquid chromatography (HPLC) on a TSK G3000 SW column (Tosoh, Tokyo, Japan). Protein was detected at an absorbance of 280 nm. An outline detector Model 170 (Beckman Coulter, Inc., Fullerton, CA, USA) was connected to monitor the radioactivity of each fraction. 
 In addition, 188Re-labeled C218 was incubated with serum of a normal adult at 37deg.C, and aliquots were taken at 24, 48, and 96 h, and then analyzed by HPLC. 
 As a control, the intact MAb C218 was labeled with 188Re without 2IT modification as previously reported with stannous tartrate as a reduction agent.10 The specific activity was 202 MBq/mg. This non-modified 188Re-labeled C218 was incubated with serum and analyzed by HPLC. 
Affinity and immunoreactivity analysis 
MAbs C218 and NK1NBL1 labeled with 125I were incubated at different antibody concentrations with 1 x 107 NCI-H69 cells in 5.7 x 46 mm microcentrifuge tubes for 1 h at 4deg.C. After centrifugation at 1,500 x g, the tubes were washed with saline, and the pellets were taken by cutting the bottoms of the tubes. The radioactivity bound to the cells was counted in a well-type gamma counter. The binding affinity constant was calculated by means of a Scatchard analysis.21 
 To compare 188Re- and 125I-labeled C218 and NK1NBL1 in binding to NCI-H69 cells, 14 ng of radiolabeled C218 or NK1NBL1 was incubated with increasing concentrations of cells for 1 h at 4deg.C. Specific binding to cells was calculated by subtracting the nonspecific binding in the tubes in which 10 ug of unlabeled MAb was added. The immunoreactive fraction was determined by linear extrapolation to conditions representing infinite antigen excess.22 
Biodistribution studies 
Five groups of mice (5/group) bearing NCI-H69 xenografts per time point were given 296 kBq of 188Re-labeled C218 and 111 kBq of 125I-labeled C218 at the same time via a lateral tail vein, while other 5 groups were given 188Re-labeled NK1NBL1 and 125I-labeled NK1NBL1, and the other 5 groups were given with 188Re-labeled OC125 and 125I-labeled OC125. The mice were sacrificed at 1, 5, 24, 48, and 96 h after injection. The 188Re radioactivity of tumors and selected organs was determined with a well-type gamma counter, and the 125I radioactivity was measured after 10 half-lives of 188Re. The results were expressed as a percentage of the injected dose per g tissue (%ID/g) and as tumor-to-tissue ratio values of the radioactivity concentration. The average weight of the xenografts used in this study (n = 75) was 0.38 +- 0.21 g and there was no significant difference among groups. The "Principles of laboratory animal care" (NIH publication No. 86-23, revised 1985) were followed in animal experiments. 
Statistical analysis 
The biodistribution data for 188Re-labeled MAbs and their 125I-labeled counterparts were compared by Student's t-test (p < 0.05). 
RESULTS 
On HPLC, a peak of protein-bound radioactivity was observed only at the fraction of intact antibody for 2IT-modified 188Re-labeled C218 and NK1NBL1, showing that they were not fragmented but remained intact. The in vitro stability test showed that 6%, 11%, and 22% of radioactivity became unbound from protein at 24, 48, and 96 h, respectively, for 2IT-modified 188Re-labeled C218 versus 10%, 28%, and 42%, respectively, for non-modified 188Re-labeled C218. 
 The affinity was determined to be Ka = 7.0 x 107 M-1 for C218, and Ka = 5.2 x 107 M-1 for NK1NBL1 by Scatchard analysis. The binding sites were 5.5 x 105 per cell. The cell binding assay demonstrated binding of 188Re-labeled C218 and NK1NBL1 and 125I-labeled counterparts to NCI-H69 cells (Fig. 2). Immunoreactivity was estimated to be 56% and 53% for 125I-labeled and 188Re-labeled C218, 45% and 44% for 125I-labeled and 188Re-labeled NK1NBL1, respectively. 
 Biodistributions of 125I-labeled C218, NK1NBL1, and OC125 are shown in Figure 3 as well as those of 188Re-labeled C218, NK1NBL1 and OC125 in Figure 4. Radiolabeled C218 and NK1NBL1 showed signs of more specific accumulation in the tumor than radiolabeled OC125. 
 Although 188Re-labeled C218 and NK1NBL1 showed signs of lower absolute tumor accumulation than 125I-labeled counterparts, they had higher tumor-to-blood ratios. Moreover 188Re-labeled NK1NBL1 had a higher tumor-to-lung ratio than 125I-labeled NK1NBL1 (Tables 1 and 2). 
DISCUSSION 
MAbs directly labeled with 188Re have successfully localized malignant tumors.9,10 Griffiths et al. reported that in a mouse model of human colorectal carcinoma LS174T, 188Re-Mu-9 and 131I-Mu-9 had tumor-to-blood ratios of 2.91 +- 0.41 and 1.56 +- 0.10, respectively, at 72 h.9 Hosono et al. showed that in mice bearing human colorectal tumor LoVo, 188Re-labeled B72.3 had a tumor-to-blood ratio of 2.04 +- 0.44 at 96 h whereas 125I-labeled B72.3 had a value of 1.05 +- 0.28.10
 In direct labeling methods where the endogenous complexing groups on antibodies are used, antibodies may be impaired due to splitting of disulfide bonds. Nevertheless, direct methods are simple to conduct, and require minimal antibody manipulation, which is a great advantage for clinical use.
 For labeling of antibodies with 99mTc, a modification of MAbs with 2IT, which provides thiol reactive positions, has been reported to be effective in maintaining the integrity of intact IgG or F(ab')2.14,15 
 In this study, in vitro stability was improved with the 2IT modification, as 89% of radioactivity remained bound to the modified antibody at 48 h in comparison with 58% for the non-modified antibody. Griffiths et al. also reported that 66% of radioactivity remained bound in vitro at 48 h for an antibody directly labeled with 188Re.9 
 In terms of in vivo stability, 188Re-labeled MAbs demonstrated faster clearance from the normal tissues and tumors than 125I-labeled MAbs. It is likely that 188Re-labeled MAbs are metabolized in vivo resulting in rapid washout of radioactivity, but further studies are needed to elucidate the metabolism of 188Re-labeled MAbs.8
 Bifunctional chelates are another approach which should enhance the targeting ability of rhenium-labeled antibodies.11,12 Goldrosen et al. reported that NR-LU-10 MAb formed a stable conjugation with 186Re by using a tetrafluorophenyl-activated ester derivative of the triamide thiolate as a chelate, and showed blood, liver, kidney and tumor accumulation of 2.2, 0.3, 0.2, and 6.7%ID/g, respectively, and a tumor-to-blood ratio of 3 at 72 h in mice bearing human colon carcinoma.11 Beaumier et al. reported that the same 186Re-labeled NR-LU-10 MAb demonstrated tumor-to-blood ratios of 0.7 and 1.3 at 48 and 120 h.2 
 In this study, 188Re-labeled derivatized NK1NBL1 achieved good tumor-to-blood ratios, 4.0 and 6.8, at 48 and 96 h, comparable to chelate methods. A limitation of this study was the relatively low tumor-to-normal tissue ratios, which may be attributable to low affinity of the MAbs for the antigen. A multistep targeting technique called "Affinity Enhancement System (AES)" with a bispecific antibody and a radiolabeled hapten can reportedly increase tumor-to-normal tissue ratios even if low-affinity MAbs are used.23,24 As a matter of fact, the MAb NK1NBL1 used in the present study has demonstrated higher tumor-to-normal tissue contrast when applied to the AES technique.25 
 Radiolabeled NK1NBL1 had a higher tumor uptake than radiolabeled C218 in this study, whereas the affinity constant of NK1NBL1 Ka = 5.2 x 107 M-1 was lower than Ka = 7.0 x 107 M-1 for C218. Pharmacokinetics of C218 and NK1NBL1 in the blood were similar. A hypothesis proposed by Fujimori et al. offers a suggestion concerning this discrepancy. They demonstrated that the average antibody concentration in the tumors does not increase linearly with antibody affinity because macromolecular ligands could be prevented from penetrating tumors by their successful binding to the target receptor.26 This could in part explain the difference between MAbs C218 and NK1NBL1 in tumor localization as a function of antibody affinity, although many of the factors that determine antibody distribution are still not understood.
 In conclusion, the anti-NCAM MAbs directly labeled with 188Re by means of 2IT derivatization seems effective in targeting SCLC tumors. 
ACKNOWLEDGMENTS 
The authors are grateful to the Government of France for the fellowship offered to Makoto Hosono. They thank Professor Hans J. Biersack, Bonn, Germany for his academic advice. ORNL is operated for the U.S. Department of Energy (DOE) under contract with the Lockheed Martin Energy Research Corporation. This study was supported in part by grants-in-aid from the Ministry of Education (No. 11670911), Japan. 
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