ORIGINAL ARTICLE 

Annals of Nuclear Medicine Vol. 15, No. 3, 199-202, 2001 

Experimental radioimmunotherapy with 186-Re-MAG3-A7 anti-colorectal cancer monoclonal antibody: Comparison with 131-I-counterpart 

Seigo KINUYA,* Kunihiko YOKOYAMA,* Katsutoshi KOBAYASHI,** Shoji MOTOISHI,** Katsuyuki ONOMA,** Naoto WATANABE,*** Noriyuki SHUKE,**** Hisashi BUNKO,***** Takatoshi MICHIGISHl* and Norihisa TONAMI* 

*Department of Nuclear Medicine, Kanazawa University School of Medicine 

**Production Division, Department of Research Reactor, Division of Radioisotopes, Japan Atomic Energy Research Institute 

***Department of Radiology, Toyama Medical and Pharmaceutical University 

****Department of Radiology, Asahikawa Medical College 

*****Medical Informatics, Kanazawa University Hospital 

A murine IgGl against a Mr 45 kD tumor-associated glycoprotein in human colorectal cancer, A7, was radiolabeled with 186-Re by a chelating method with a mercaptoacetyltriglycine (MAG3). Its specific activity was 119 MBq/mg, which would be high enough for a therapeutic purpose, and its immunoreactivity was preserved well as was 131-I-A7 labeled by the chloramine-T method. Growth of human colon cancer xenografts, 9.14+-0.44 mm in diameter, in nude mice was significantly suppressed by an intravenous dose of 4.48 MBq of 186-Re-A7. The therapeutic outcome with 186-Re-A7 was better than that with 4.63 MBq of 131-I-A7. Toxicity of treatments assessed by body weight change was similar with both conjugates. These results are likely caused by the tumor size and more favorable physical properties of 186-Re than those of 131-I. 

Key words: radioimmunotherapy, 186-Re, colon cancer xenograft 

INTRODUCTION 

131-I is the radionuclide that has been most widely used to label monoclonal antibody (MAb) for radioimmunotherapy (RIT).1,2 One of major disadvantages of 131-I is high energy remission, 364 keV, that is not ideal for gamma detection and exposes patients to unnecessary radiation 186-Re appears to be a suitable radionuclide for RIT with its appropriate physical half-life of 3.7 days that is long enough for MAb to localize tumors and short enough to minimize toxicity in the whole body.

Received November 20, 2000, revision accepted February 1 , 2001 . 

For reprint contact: Seigo Kinuya, M.D., Department of Nuclear Medicine, Kanazawa University School of Medicine, l3-1 Takaramachi, Kanazawa, Ishikawa 920-8640, JAPAN. 

E-mail : kinuya@med.kanazawa-u.ac.jp

Abundant intermediate energy p emission (71 % of 1.07 MeV and 21 % of 0.94 MeV) is comparable to 131-I, and y emission of 137 keV (9%) that is suitable for external detection with gamma cameras, which may provide more accurate tissue absorbed radiation dose estimation than with 131-I, and produces a less nonspecific radiation dose than 131-I. 

186-Re has similar chemical properties to 99m-Tc. Although 
99m-Tc-MAb is now widely used for radioimmunoscintigraphy (RIS),3,4 radiolabeling is performed by a direct labeling method that is not ideal for 186-Re because of the instability of directly labeled 186-Re-MAb,5 so that indirect methods with ligands such as N2S2, N2S4 and N3S compounds have been investigated.6-9 Among these, a prechelating labeling method with S-benzoylmercapto-acetyltriglycine (MAG3), an N3S ligand, appears to be a good choice because of its in vivo stability and possible high specific activity of labeled MAb.8 

In this study of a mouse model xenografted with human colon cancer cells, we sought to determine the efficacy of RIT of 186-Re-MAG3-MAb. This study was performed as a part of the Working Group on Radioactive Rhenium supported by the Consultative Committee of Research on Radioisotopes and the Subcommittee for Production and Radiolabeling in the Japan Atomic Energy Research Institute. 

MATERIALS AND METHODS 

A7, an IgG1 murine MAb recognizing Mr 45,000 tumorassociated glycoprotein of colorectal cancer, was used.10 186Re-perrhenate (186ReO4-) was produced by 185Re(n,r) reaction (Japan Atomic Energy Research Institute, Tokaimura, Japan) at a specific activity of 19.0 TBq/g, chelated with S-benzoyl-mercaptoacetyltriglycine (MAG3) (a gift from Dr. Yasushi Arano) and conjugated to A7.8 Briefly, the mixture of 186ReO4-, SnC12 and S-benzoyl-MAG3 at the molar ratios of 2.3 : 1 for S-benzoyl-MAG3 : Re and 8.0 : 1 for Sn2+ : Re was heated under an N2 stream, resulting in 186Re-MAG3, which was conjugated to A7 after esterification with 2,3,5,6-tetrafluorophenol (TFP) (Nacalai Tesque, Kyoto). 186Re-MAG3-A7 was then purified on a PD10 column (Pharmacia LKB Biotechnology, Uppsala, Sweden) with 5 mg/ml ascorbic acid as an eluant to prevent the radiolysis of the MAb. Immunoreactivity of 186Re-MAG3-A7 was determined with I .6 x 105 to 3 x 106 of LS 1 80 human colon carcinoma cells (American Type Culture Collection. Rockville. MD, USA) as described by Lindmo et al.1 1 The labeled MAb was sterilized by means of a filter (Millex-GV, 0.22 mm; Millipore, Bedford. MA, USA) prior to further experiments. 

Animal studies were performed in compliance with the regulations of our institution. LS 180 cells were grown in DMEM medium (Nissui Seiyaku, Tokyo), harvested with 0.1% trypsin, and then 5 x 106 of cells were subcutaneously xenografted into the thigh of Balb/c nu/nu mice (female, 20 g; NlNOX Labo Supply Inc.. Ishikawa). Tumor volume (mm3) was calculated as length (mm) x width (mm)2 x 0.5, and expressed as the ratio of volume to the volume on day 0 (the day of starting the treatment). Tumor volume on day 0 was 376+-46 mm3 and the diameter was 9.14+-0.44 mm. The tumoricidal activity of a dose of 4.48 MBq (121 uCi) of 186Re-A7 was determined (n = 9). As a comparison, the therapeutic effect of 4.63 MBq ( 125 uCi) of 13lI-A7 Iabeled by the chloramine-T method was observed in the same model (n = 8). Tumor growth in non-treated mice was also observed as a reference (n = 5). Toxicity of the treatment was assessed by body weight loss of the animals. 

Absorbed radiation dose in tissue with 186Re-A7 was estimated under the assumption that 186Re-A7 would show similar biodistribution to 131I-A7: with the labeling condition yielding an appropriate conjugation ratio of 186Re-MAG3 to MAb, 186Re-MAbs was cleared from the circulation and accumulated into tumors similarly to 125I-MAbs, and distribution of 186Re-MAbs in normal tissue did not vary from that of 125I-MAbs with some exceptions in gastric accumulation and their excretion routes. 12,13 These were demonstrated in both normal mice and tumorbearing mice. In the estimation, we used the previous biodistribution data obtained with 125I-A7, 14 and the physical half lives of 186Re and 131I were adapted to the data to obtain effective cumulative radioactivity within tissue for 186Re and 131I. Absorbed radiation dose was estimated by the formula: DB = uCi x h x g-1 x E, where E of 131I 0.3985 and E of 186Re = 0.73.13 The contribution of r emission was neglected in the calculation. 

RESULTS 
The efficiency of 186Re-MAG3-TFP production was 74%, and 60% of 186Re-MAG3-TFP was conjugated to A7. The specific activity of 186Re-A7 was 119 MBq/mg, and its immunoreactivity at infinite antigen excess was 72%. Those of 131I-A7 were 140 MBq/mg and 71 %.  

RIT with 186Re-A7 significantly suppressed the growth of xenografts as compared to no treatment (Fig. 1 and Table 1 ). A dose of 4.48 MBq of 186Re-A7 showed better tumor suppression than did a dose of 4.63 MBq of 131I-A7. Maximum body weight loss was similar with both conjugates at this dose level (Table 1), but the loss with 131I-A7 tended to appear later and persist longer than that with 186Re-A7: a nadir on day 6 with 186Re-A7 and on day 12 with 1311-A7. No mouse died from the treatment during the observation period. 

Estimated tissue absorbed radiation doses are shown in Table 2. The absorbed radiation dose caused by p emissions to the tumor with a dose of 4.48 MBq of 186Re-A7 was 1.67-fold greater than that with 4.63 MBq of 131I-A7. Doses absorbed by normal tissue from p emissions were approximately 1.5-fold greater with 186Re-A7. 

DISCUSSION 

A7 MAb was able to be labeled with 186Re-MAG3 at sufficiently high specific activity for a therapeutic purpose, and its immunoreactivity was well preserved. We found significant tumoricidal effect of 186Re-A7 in vivo, and 186Re-A7 produced better tumor response than did 131I-A7 at the similar dose level. Estimation of the tissue absorbed radiation dose indicates that 186Re-A7 produced a much greater tumor dose than 131I-A7, which would be the major reason for the better outcome with 186Re-A7. The size of tumors may be another factor in the more pronounced tumor suppression with 186Re-A7 than 131I-A7. The efficacy of RIT is affected by the properties of the radionuclide labeled to MAbs, and a mathematical model assuming uniform radionuclide distribution in tumors indicates that the optimal cure tumor size for B-particles of 186Re (71% of 1.07 MeV and 21 % of 0.94 MeV) is 7.0-12.0 mm in diameter in contrast to 2.6-5.0 mm for 131I (86% of 0.606 MeV and 13% of 0.336 MeV).15 Kievit et al 13 reported the slight superiority of 131I MAb to 186Re-MAb in 5.0-7.0 mm ovarian cancer xenografts delivered with the equal tumor absorbed dose by two conjugates, concluding that the tumor size contributed to producing these findings. In contrast, the diameter of tumors used in this study was 9.14 +- 0.44 mm, being within the optimal cure range for 186Re. In current clinical settings, patients with recurrent lesions and metastatic lesions are candidates for RIT. In general, the minimal size of a tumor that is detectable with imaging methods is around 1 cm, which is within the suitable range for the B-particles of 186Re. In addition, to treating larger tumors, the so-called cross-fire effect from radiolabeled MAbs heterogeneously distributed within tumors may be more significant with B-particles of 186Re than those of 1311. These several factors suggest the superiority of 186Re-A7 to 131I-A7 as an RIT compound. 

Body weight was monitored to assess the toxicity of treatments, indicating that maximum body weight loss in the group treated with a dose of 4.48 MBq of 186Re-A7 was similar to that with 4.63 MBq of 131I-A7. In contrast, absorbed radiation doses within normal tissues including whole body doses were approximately 1.5-fold greater with 186Re-A7 than with 131I-A7 at these doses. We neglected the contribution of y emissions in the estimation of tissue radiation dosimetry, and abundant high energy y emission of 364 keV of 131I may have produced a considerable whole body radiation dose, as compared with the lower energy r-emission of 186Re, so that the actual whole body radiation dose with 131I-A7 is likely to be closer to that with 186Re-A7 than shown in Table 2, which made the toxicity similar with both conjugates. r-emissions would contribute to the whole body dose in human subjects more significantly than in small animals, suggesting that the advantage of 186Re-A7 over 131I-A7 would be greater in human subjects than in animals with regard to toxicity. The different profile in terms of the duration of body weight loss with two conjugates may depend on the difference between the physical half-lives of these radionuclides. 

In conclusion, RIT with 186Re-A7 suppressed the growth of colon cancer xenografts more effectively than that with 131I-A7 at a similar dose level. They were equally toxic when assessed by body weight change. These results are likely to be caused by the tumor size treated in this study and the more favorable physical properties of 186Re than those of 131I. 

ACKNOWLEDGMENTS 

We thank former Professor Toshio Takahashi and Dr. Toshiharu Yamaguchi, First Department of Surgery, Kyoto Prefectural University of Medicine, for providing A7 MAb, and Dr. Yasushi Arano, Faculty of Pharmaceutical Sciences, Kyoto University (currently Professor of Faculty of Pharmaceutical Sciences, Chiba University), for providing S-benzoyl-MAG3. 

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