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Medulloblastomas are the most common type of primitive neuroectodermal tumours observed in the central nervous system of children. In spite of recent advances in therapy, including microsurgery, combined chemotherapy and radiotherapy, the longterm prognosis for patients with these tumours is not satisfactory (Evans et al, 1990), pointing to a need for more specific and efficient therapies. Antibodies that recognize medulloblastoma cells are expected to act as diagnostic and therapeutic reagents. Therefore, we attempted to produce a medulloblastoma-specific monoclonal antibody (MAb). Immunization of mice with ONS-76 medulloblastoma cells (Tamura et al, 1989) successfully produced the antibodies referred to as ONS-M21 MAb (Moriuchi et al, 1993). ONS-M21 MAb reacted with a surface antigen of most glioma and medulloblastoma cell lines, and it also reacted with surgical specimens of gliomas and medulloblastomas. On the other hand, this MAb did not show cross-reactivity with peripheral blood cells or normal brain tissue. However, mouse antibodies are highly immunogenic in humans, and, hence, their clinical use may be limited. We genetically engineered a recombinant, humanized version of ONS-M21 MAb (hONS-M21 Ab) by means of CDR grafting (Ohtomo et al, 1995) in order to eliminate immunogenicity. This humanized Ab competed with ONS-M21 MAb for binding to glioma cells. In addition, this Ab, when labelled with an isotope, was capable of depicting tumour location by an autoradiographic technique. Thus, humanized Ab possesses various advantages that make it attractive for clinical applications.As the target molecule of ONS-M21 remains unknown, identification of this antigen is a necessary step before any clinical use of this antibody can be attempted. In the present study, we purified ONS-M21 antigen from ONS-76 cell lysates by means of an Abbinding affinity column, analysed the amino acid sequences of the ONS-M21 binding protein and identified the target molecule of ONS-M21 MAb. In addition, the mRNA level of this antigen was analysed by reverse transcription-polymerase chain reaction (RT-PCR) in surgical specimens of gliomas and medulloblastomas. MATERIALS AND METHODS Cell culture conditions and preparation of the monoclonal antibodyThe human medulloblastoma cell line ONS-76 was established in our laboratory (Tamura et al, 1989; Institution for fermentation, Osaka, No. 50355), maintained in Dulbecco's modified Eagle medium (DMEM) with 10% heat-inactivated fetal bovine serum (FBS) and 50 µg ml -1 gentamycin, and incubated in a humidified 10% carbon dioxide-in-air atmosphere at 37°C. ONS-76 cells were collected using a cell scraper and washed with cold Trisbuffered saline (TBS; 10 mmol l -1 Tris-Cl, 150 mmol l -1 NaCl, pH 8.0). The cells were resuspended in a detergent solution (TBS with 0.5% Tween-20 and 1 mg ml -1 p-amidinophenyl methanesulphonyl fluoride hydrochloride) at a concentration of 10 6 cells ml -1 , and then shaken for 2 h at 4°C to elute the cell extract. After centrifuging (3000 r.p.m., 20 min), the supernata...

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Intravenous gene transfer using recombinant retroviruses tends to suffer from a low infectious viral titer when conducted in vivo. This is, in part, caused by complement‐mediated proteolytic inactivation of the retrovirus in human serum. However, if the retroviruses were directly injected into the brain, they might not be inactivated. Supernatant from amphotropic retrovirus‐producing cells harboring the BAG vectors was incubated with sera or cerebrospinal fluid (CSF) of patients with gliomas or unrelated disorders. The retroviruses were severely inactivated in sera. However, no such inactivation was noted in CSF or fluid from the tumor bed of glioma patients. These data suggest that gene transfer using recombinant retroviruses could be done into the cavity after removal of the tumor in glioma patients.

The intracranial migration of transduced glioma cells was retroviruses originating from the high-titer retrovirus-proinvestigated in order to improve the treatment of malignant ducing cells. Besides the importance of using a high-titer glioma by gene therapy using retroviral vectors. In this retroviral vector system, our results also indicate that the study, about half the volume of the tumor mass could be implantation site of the virus-producing cells and the intertransduced in 14 days after only a single implantation of val between the implantation of the virus-producing cells 3 × 10 5 retrovirus-producing cells into a tumor mass with a and the subsequent administration of ganciclovir are diameter of 5 mm. Moreover, we were able to follow the important factors for the efficient killing of glioma cells. migration of glioma cells transduced by the lacZ-harboring