Makoto Shirato

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.

A human/mouse chimeric monoclonal antibody against CA125 for radioimmunoimaging of ovarian cancer

Murine monoclonal antibody 196-14 recognizes the ovarian-cancer-associated antigen CA 125, but the epitope it recognizes is different from that of monoclonal antibody OC125. We developed a human/mouse chimeric 196-14 using the variable regions of the murine 196-14 and human heavy-chain (γl) and light-chain (κ) constant regions. Cell binding and competitive inhibition assays using chimeric 196-14 labeled with125I,111In or99mTc demonstrated that the in vitro immunoreactivity of the chimeric antibody was identical to that of the parental murine monoclonal antibody. However, in mice bearing human ovarian cancer xenografts, the clearance from blood was faster and absolute levels of accumulation in the tumor were lower for the125I-labeled or99mTc-labeled chimeric antibody than for the murine antibody labeled with the corresponding radionuclides. The tumor-to-blood radioactivity ratio was not significantly different between the chimeric antibody and the murine antibody, regardless of the radionuclide used for labeling. Chimeric antibody 196-14 labeled with131I,111In or99mTc is promising for the radioimmunoimaging of ovarian 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.

Scintigraphic detection of xenografted tumors producing human basic fibroblast growth factor

A murine monoclonal antibody 3H3 recognizes the basic fibroblast growth factor (FGF) and inhibits the growth of human glioblastoma cells both in vitro and in vivo. We studied the potential of a scintigraphic technique using the 3H3 antibody to detect tumors that produce basic FGF.125I- and111In-labeled 3H3 bound to U87MG human glioblastoma cells in vitro. U87MG cells were inoculated subcutaneously into nude mice. After development of the tumor, radiolabeled 3H3 was injected into the subcutaneous space surrounding the tumor. A high level of radioactivity from 3H3 was retained at the tumor, whereas an irrelevant antibody cleared rapidly from the injected site. Radiolabeled 3H3 was not retained in tumors that did not produce basic FGF. Scintigraphic detection of tumors expressing basic FGF would be valuable for the therapeutic application of the antibody.

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.

Immunoscintigraphy of colorectal cancer using 111In-labeled monoclonal antibody to mucin.

A murine monoclonal antibody MLS102 recognizes sialosyl-Tn antigen in mucin and immunohistochemically reacts with more than 80% of colorectal cancer tissues. The purpose of this study was to assess the usefulness of this monoclonal antibody for the immunoscintigraphy of colorectal cancer. Planar and SPECT images were obtained on day 2 or day 3 after injection of 2 mg and 74 MBq111In-labeled MLS102 antibody into 17 patients with colorectal cancer. Nine of 11 primary tumors and 4 of 6 locally recurrent tumors were detected. Positive images were obtained in all tumors larger than 4.5×2.7 cm. Three tumors of less than 2.5 cm and 1 recurrent tumor, which was missed by other imaging modalities, were negative. There were no adverse reactions. Human anti-(mouse Ig) antibody developed in 4 patients. Although improvement of detectability for smaller tumors needs to be pursued, the antibody MLS102 is potentially promising for use in immunoscintigraphy of colorectal cancer.

Pentavalent technetium-99m dimercaptosuccinic acid SPECT of metastatic renal cell carcinoma in the scalp and in the brain.

The authors report marked accumulation of pentavalent technetium-99m dimercaptosuccinic acid in metastatic tumors from a renal cell carcinoma in the brain and the scalp on brain SPECT images.