Hisataka Kobayashi

Comparison of the Chase Effects of Avidin, Streptavidin, Neutravidin, and Avidin‐Ferritin on a Radiolabeled Biotinylated Anti‐tumor Monoclonal Antibody

Injection of avidin can decrease the background radioactivity due to a radiolabeled biotinylated monoclonal antibody. We compared the chase effects of avidin, streptavidin, neutravidin, and avidin‐conjugated ferritin on a radiolabeled antitumor monoclonal antibody in tumor‐bearing nude mice. A radioiodine‐labeled biotinylated monoclonal antibody (OST7) was administered to athymic mice bearing osteogenic sarcomas. After 24 h, an avidin, streptavidin, neutravidin or avidin‐conjugated ferritin chaser was intravenously injected into the mice. At 2 h after the chase, the biodistribution of the radiolabeled monoclonal antibody was determined. Clearance from the blood was dose‐dependently accelerated by avidin and its effect was 10‐fold stronger than that of neutravidin or avidinferritin. Streptavidin did not promote clearance of the biotinylated antibody. Avidin was the most effective chasing agent for improving the biodistribution of the radiolabeled biotinylated monoclonal antibody among the four avidin derivatives tested.

In vivo instability of reduction-mediated 99mTc-labeled monoclonal antibody

A murine monoclonal antibody that reacts with human osteogenic sarcoma (OST7) was reduced and directly labeled with 99mTc without any loss of immunoreactivity. No fragmentation of the antibody was detected by high performance liquid chromatography after the labeling. However, SDS-PAGE analysis of the labeled antibody demonstrated the presence of low molecular weight species. Although more than 95% of the radioactivity remained bound at the antibody after incubation with human serum for 24 h, 99mTc-labeled OST7 was cleared faster from the circulation than 125I-labeled OST7 or 111In-labeled OST7 in mice. Urinary and fecal excretion of 99mTc were higher than those of 125I. When the 125I-labeled antibody was dual-labeled with 99mTc, the blood clearance of 99mTc was faster than that of 125I, suggesting release of 99mTc from the antibody in vivo. 99mTc-labeled OST7, however, gave a higher tumor-to-blood ratio than 125I- or 111In-labeled OST7 in mice bearing human osteogenic sarcoma. The 99mTc-labeled antibody prepared by the direct method was unstable in vivo, but retained a good tumor targeting ability.

Radioimmunoimaging of colon cancer xenografts with anti-Tn monoclonal antibody

Tn antigen is a glycosylated tumor associated antigen and a murine monoclonal antibody, MLS128, has been identified to react with it. The potential of MLS128 for the radioimmunoimaging of colorectal cancer was studied. MLS128 was labeled with radioiodine by the chloramine-T method or indium-111 (111In) by using isothiocyanatobenzyl EDTA, and was injected into nude mice bearing human colon cancer xenografts. Radiolabeled MLS128 showed a high and specific localization in xenografted tumor. At 48 h after injection, the %ID/g of 125I-labeled MLS128 in the tumor was 34.69, whereas that of isotype matched control antibody, FLOPC21, was 5.58 and the tumor-to-nontumor radioactivity ratios of 125I-labeled MLS128 reached to 4.56, 17.84 and 23.62 for the blood, liver and bone, respectively. 111In-labeled MLS128 showed similar results. High accumulation of MLS128 in xenografted tumors suggested that the monoclonal antibody MLS128 is promising for radioimmunoimaging of colorectal cancer.

Scintigraphic detection of neural-cell-derived small-cell lung cancer using glioma-specific antibody

Radiolabeled GA-17, a murine monoclonal antibody that reacts specifically with glioma cells, bound to a small-cell lung cancer (SCLC) cell line NCI-H69 derived from neural cells, both in vitro and in vivo. The affinity constant of GA-17 F (ab′)2 fragment binding to NCI-H69 was 1.02×108/M while that to the glioma cell line U87MG was 1.22×108/M. Iodine-125-labeled GA-17 F (ab′)2 fragments injected i.v. localized well in NCI-H69 cells xenografted in nude mice. The percentage of the injected dose per gram accumulated in the xenografted tumor was 6.87±1.34%g−1 (mean±SD,n=5) 24 h after injection. On the other hand, control monoclonal F (ab′)2 fragments accumulated in the xenografted tumor at 0.75±0.30%g−1. The tumor-to-blood ratio was 1.8 for NCI-H69, while that of control F (ab′)2 was 0.60. In conclusion, the radiolabeled GA-17 F (ab′)2 fragment is expected to be useful clinically to visualize the small-cell lung cancer and in radioimmunotherapy.

Construction of Immunoradiometric Assay for Circulating c‐erbB‐2 Protooncogene Product in Advanced Breast Cancer Patients

The human c‐erbB‐2 protooncogene product (erbB‐2 protein) is a 185 kilodalton glycoprotein closely related to epidermal growth factor receptor protein. In this study, we measured the concentration of circulating erbB‐2 protein in cancer patients by means of a new immunoradiometric assay (IRMA). Two monoclonal antibodies (MoAbs), SV2‐61γ and 6G10, recognize erbB‐2 protein but bind to separate epitopes. SV2‐61γ was used as an immunoadsorbent and 6G10 as an 125I‐labeled probe. A serum was considered positive for erbB‐2 protein if the percent binding exceeded the mean of the normal group by more than 3 standard deviations. Eleven of 21 patients with advanced breast cancer and 1 of 15 with advanced gastric cancer were positive. Serum erbB‐2 protein levels correlated well with the therapy and the status of the patients with breast cancer. On the contrary, all patients with advanced colon, ovarian, or pancreatic cancers, showed levels below the cut‐off value. These results suggest that circulating erbB‐2 protein can be measured using the newly constructed IRMA. Since c‐erbB‐2 protooncogene amplification and overexpression are accepted as a good marker of aggressiveness, relapsing potency, and poor prognosis, this IRMA should be a promising tool with which to help manage breast cancer patients.

Kinetics of Platelets in Dogs with Thrombocytopenia Induced by Antiglycoprotein IIb/IIIa Receptor Monoclonal Antibody

To experimentally assess the kinetics of platelets in thrombocytopenia, we constructed a canine model using 111In-oxine labeled autologous platelets and an intact antiplatelet monoclonal antibody (MAb) NNKY2-11 (IgG2a). With the infusion of radiolabeled autologous platelets into dogs, the peripheral platelet count and blood radioactivity level were examined, and the radioactivity in the liver, spleen and heart was determined with scintigraphic analysis. Thereafter, i.v. injection of 100 micrograms/kg of NNKY2-11 had no effect on platelet counts or the biodistribution of radiolabeled platelets. However, 200 and 300 micrograms/kg of MAb reduced the platelets, and the radioactivity of the liver and spleen augmented clearly after injection of MAb. Platelet radioactivity in serum, which had decreased after MAb infusion, did not recover, even when peripheral platelet counts returned to the normal levels, indicating that these new platelets might be derived from the platelet-storage pool or new thrombocytogenesis. This model of antiplatelet MAb induced thrombocytopenia seems to be useful for analyzing the kinetics of platelets in thrombocytopenia.

Effect of circulating antigen on immunoscintigraphy of ovarian cancer patients using Anti-CA125 monoclonal antibody

The murine monoclonal antibody (mAb) 145–9 recognizes an epitope present on CA125 but different from the epitope defined by the mAb OC125. To evaluate the clinical usefulness of the 145–9 antibody, immunoscintigraphy was performed in ovarian cancer patients and the effect of circulating CA125 on tumor imaging was investigated. Two milligrams (74 MBq) of 111In‐labeled 145–9 was injected intravenously into 11 patients with ovarian cancer. Pre‐injection serum CA125 concentrations were between 166 U/ml and 7414 U/ml. Tumors were visualized in 10 of 11 patients. In two patients, lymph nodes that were not detected by other imaging modalities but were clinically suspected as metastases were visualized. There was no correlation between serum CA125 level and antibody uptake in the tumors. Immune complexes between the antibody and circulating antigen were observed in sera of all the patients, but the fraction of radioactivity in complex form did not correlate well with serum CA125 levels. The immune complexes survived in the circulation and the circulating radiolabel, including immune complexes, was still bound to solid‐phase CA125. The plasma clearance rate and hepatic uptake of the antibody were not significantly affected by circulating CA125. In conclusion, the antibody 145–9 formed complexes with CA125 in vivo but this did not compromise the outcome of antibody imaging. The antibody 145–9 can be used in immunoscintigraphy of ovarian cancer irrespective of serum CA125 level.

Changes in CA125 release and surface expression caused by drugs in uterine cervix adenocarcinoma cells

The effect of drugs on the release of CA125 antigen and the binding of anti-CA125 mono-clonal antibody (MoAb) to malignant cells was evaluatedin vitro. TMCC-1, uterine cervical adenocarcinoma cells, were exposed to dexamethasone (DEX), sodiumn-butyrate (NaB), dibutyryl cyclic AMP (dbcAMP), retinoic acid (RA), calcitriol (VD3), and interferon-γ (IFN-γ). NaB, RA and VD3 increased CA125 release per cell and125I-labeled anti-CA125 MoAb binding to the cells. DEX also increased the125I-labeled anti-CA125 MoAb binding to the cells, and CA125 antigen release per cell was also slightly increased. IFN-γ suppressed both CA125 release and125I-labeled MoAb binding. A combination of DEX, VD3 and RA and increased the binding of MoAb to TMCC-1 cells, but the amount of bound MoAb was not significantly different from that obtained by single drug treatment. DbcAMP had no significant effect on enhancing MoAb binding. Drugs can increase the binding of anti-CA125 MoAb to malignant cells and they may be applied to increase the tumor uptake of radiolabeled MoAbsin vivo.

Inflammation-seeking scintigraphy with radiolabeled biotinylated polyclonal IgG followed by the injection of avidin chase

We tried to apply the avidin chasing system to the inflammation-seeking scintigraphy using radiolabeled nonspecific polyclonal IgG. We studied the pharmacokinetics of technetium-99m and iodine-125-labeled biotinylated murine polyclonal IgG followed by an avidin chase injection in model mice with inflammatory foci. Avidin chase decreased the circulating radioactivity of 99mTc and 125I, which was a major problem for inflammation-seeking scintigraphy using radiolabeled nonspecific polyclonal IgG, to 9.3% and 19.3% of that without an avidin chase injection, respectively. Inflammation-seeking scintigraphy with the aforementioned method would be better than that with conventional method.

Radioimmunodetection of human leukemia with anti-interleukin-2 receptor antibody in severe combined immunodeficiency mice

Anti-Tac monoclonal antibody recognizes human interleukin-2 receptor, which is overexpressed in leukemic cells of most adult T-cell leukemia (ATL) patients. To examine the potency of anti-Tac for targeting of ATL, biodistributions of intravenously administered 125I- and 111In-labeled anti-Tac were examined in severe combined immunodeficiency (SCID) mice inoculated with ATL cells. Significant amounts of radiolabeled anti-Tac were found in the spleen and thymus. The trafficking of ATL cells in SCID mice was detected using 111In-oxine-labeled ATL cells. These results were coincident with the histologically confirmed infiltration of ATL cells. The radiolabeled anti-Tac seemed potent for targeting of ATL.