Journalof Neuro-Oncology 8: 221-229, 1990. © 1990KluwerAcademic Publishers. Printedin the Netherlands. Laboratory Investigation
Radioimaging of human glioma xenografts with 123I labeled monoclonal antibody G-22 against glioma-associated antigen Jun Yoshida, Masaaki Mizuno, Itaru Inoue, Toshihiko Wakabayasi, Kenichiro Sugita, Hisao Seo ~ and Kazumi Chiba 2 Department of Neurosurgery, Nagoya University School of Medicine, Nagoya, Japan; 1Department of Endocrinology and Metabolism, Research Institute of Environmental Medicine, Nagoya, Japan; 2 Daiichi Radioisotope Laboratories, LTD, Chiba, Japan
Key words: radioimaging, monoclonal antibody, glioma, glioma-associated antigen, 123I
Summary Monoclonal antibody (MCA) G-22 is directed against a human glioma-associated surface antigen. Its availability for the radioimmunodetection of human glioma was analyzed by utilizing the xenografts in athymic mice. Nude mice with subcutaneous grafts of U251-MG or U251-SP glioma received intravenous administration of 123Ior 131Ilabeled F(ab')2 fragment or whole immunoglobulin. Results of radioimaging revealed that 123I-labeled antibody was better than the 13~I-labeled. It was also noted that administration of L23I-labeled F(ab')2 fragment of G-22 MCA enabled the imaging of human glioma xenografts weighing 80-650mg after 48 hours. When biodistribution of 123I MCA was compared between G-22 and control antibodies, the percentages of dose/g in tumors were 5.228-1.799 at 30 hours and 4.112-1.132 at 48 hours with G-22 and they were 4.164-1.248 and 0.314-0.142 with control. The tumor/blood ratio until 72 hours after injection was constantly above 1 with G-22 and less than 1 with control antibody. These results indicate the potential usefulness of G-22 MCA for the radioimmunodetection of human gliomas.
Introduction Since Pressman and Keighley [1, 2] have introduced the method of specific targeting of radiolabeled antibody to tumors, tumor imaging has been investigated in human tumor xenograft models [3, 4] as well as in human trial [5-7]. With brain tumor, Mahaley and Day  demonstrated specific localization of 131Ilabeled rabbit anti-human glioma antibody in human brain tumor, although some problems such as cross-reactivity to normal tissue or heterogeneity of antibody-specificity were recognized by the use of hetero-antibody. The introduction of monoclonal antibody (MCA) improved tumor imaging by their high specificity. Radio-labeled MCAs against tumor-as-
sociated antigens have been used for the imaging of the various tumors; colorectal carcinoma [9, 10], melanoma , osteosarcoma , ovarian carcinoma and breast cancers . For human glioma, Kemshead et al. have demonstrated positive brain tumor images after injection of 131I labeled MCA UJ13A in patients. However, later paired-labeled studies revealed that no specific antibody localization was observed with this antibody. Probably it was due to cross reactivity of the antibody to normal nervous tissues . In our laboratory, a variety of MCAs against human glioma-associated antigens was established and their clinical application for the diagnosis or the treatment has been investigated [15-19]. One of the MCAs designated as G-22 recognized a neu-
222 roectodermal antigen present in many human glioma cell lines, primarily in human glioma, but not in normal adult brain. In the present study F(ab')2 fragment of G-22 MCA was labeled with 123Iand its usefulness for the radioimmuno-detection of glioma was evaluated utilyzing human glioma xenograft models.
Materials and methods
Human glioma xenograft models Two cell lines, U251-MG and U251-SP, originally established by Pont6n et al. of the University of Upsalla from human glioma , were obtained from Memorial Sloan Keterring Cancer Institute and KMS II established by Mihara from human ependymoma was obtained from Kumamoto University . They were maintained in Eagle's minimum essential medium containing 10% fetal bovine serum, non-essential amino acids, L-glutamine, NaHCO3, streptomycin, and penicillin. All three cell lines were successfully transplanted into the subcutaneous space of female nu/nu nude mice in our laboratory. For the experiments, a tumor mass obtained from a nude mouse was cut into pieces of about 2 x 2 mm. A tumor fragment was then inoculated subcutaneously in the right abdominal region of nude mouse using a trocar needle.
U251-MG and U251-SP are positive with G-22 MCA and KMS II is negative.
Radioiodination of antibody The IgG fraction of G-22 MCA and control MCA of mouse IgG was purified from the ascite fluids by precipitation with ammonium sulfate (50% saturation) followed by chromatography on a DEAEcellulose column. To obtain F(ab')2 fragment, it was digested with pepsin at 37°C overnight. The F(ab')2 fragment was separated by chromatography on a Sephadex G-150 column. Radiolabelling of the antibody or the F(ab')2 fragment with 123I o r 131Iwas performed using a modified chloramine T technique . Free iodine was removed from the reaction mixture by filtration on Sephadex G-25. After sterilization by filtering through 0.22/xm millipore filter, the antibodies were administered to nude mice within 3-6 hours of radioiodination. The reactivity of the labeled antibody with antigen was checked by three step dot-blot ELISA as previously reported . In brief, an aliquot of purified antigen was spotted onto the nitrocellulose membrane and reacted with G-22 MCA (The first step). The bound MCA was reacted with peroxidasecoupled anti-mouse IgG (The second step). The amount of membrane bound MCA was quantitated by measuring optical density after soaking the membrane in the enzyme substrate containing Ophenylenediamine (The third step).
Monoclonal antibody A mouse MCA designated as G-22 was produced by immunization of a human glioma cell line, SKMG-4. The distribution and biochemical characterization of an antigen that react with this antibody were described in the previous papers . Briefly, the antigen is expressed in human brain of fetuses in early gestation but not in that of the adult, suggesting that the antibody recognizes neural differentiation antigen. High incidence of the expression of this antigen was observed in human glioma but not in neuroblastoma. Thus the antigen termed G-22 MCA was defined as a glioma-associated antigen. Among cell lines used in this experiment,
Imaging protocol (Table 1) Six nude mice bearing U251-SP tumor (n = 3) or KMS II (n = 3) were used for the experiment I. They were injected intravenously with 131I-G-22 IgG at a dose of 60/~Ci/10/~g/mouse. Images were obtained at day 1, 3, 5, and 7. For the experiment II another four nude mice bearing U251-SP tumor were injected with 131I-G-22 F(ab')2 fragment at the dose of 50 tzCi/10/zg/mouse and images were obtained at day 3. For the experiment III, ten nude mice bearing U251-MG tumor on the flank were divided into A (n = 6) and B (n = 4) groups. For
223 the experiment IV, fourteen nude mice bearing U251-SP tumor on the flank were also divided into A (n = 9) and B (n = 5) groups. The mice in group A from both experiment III and IV were injected with 123I-G-22 F(ab')2 fragment and those in B group with 1231control F(ab')2 fragment at a dose of 100-200/xCi/20/xg/mouse. Images were obtained 24 and 48 hours after injection in the experiment III, and 6 and 30 hours in the experiment IV with a gamma scintillation camera (Aloka OMEGA-500) using a parallel-hole collimator.
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