Yasuyoshi Miyao

An adoptive immunotherapy of patients with medulloblastoma by lymphokine-activated killer cells (LAK)

SummaryAn adoptive immunotherapy of 6 patients with medulloblastoma by lymphokine-activated killer (LAK) cells is described. They were from 2 to 9 years in age and had cerebrospinal fluid (CSF) dissemination of the tumours. All patients underwent the whole-neuraxis irradiation and chemotherapy. After the usual treatments, they were submitted to an adoptive transfer of one-haplotype identical LAK cells. The LAK cells were induced from peripheral blood lymphocytes (PBL) of their relatives with human recombinant interleukin-2 (rIL-2). 3–15×109 LAK cells were transferred intrathecally in 2–3 months. In 3 of 6 patients, neurological signs were improved and malignant cells had never been detected on CSF cytology after the adoptive immunotherapy. One among these 3 patients showed complete response in 20 months. Thus, this is an attractive approach for the treatment of medulloblastoma with CSF dissemination of the tumour which current therapeutic intervention can not cure.

Humanization of mouse ONS-M21 antibody with the aid of hybrid variable regions.

Mouse monoclonal antibody, ONS-M21, directed against human medulloblastoma cells, has been humanized by complementarity determining region (CDR) grafting. A humanized ONS-M21 VH region, comparable to the original mouse ONS-M21 VH region, was easily constructed based on framework regions (FRs) 1, 2 and 3 from human EU antibody and on FR4 from human ND antibody. Five alterations in the FRs were made at amino acids 27, 28, 29, 30 and 94 which are all part of the canonical structure for CDR1 (H1). The humanized ONS-M21 VL regions were constructed based on the FRs from human REI antibody. We first identified five amino acid residues in the FRs at positions 20, 21, 71, 73 and 87 as having a possible adverse influences on antigen binding. None of the versions with a variety of combinations at these five positions showed any bindings to antigen. In order to identify the mouse residues that must be retained in the human FRs, hybrid VL regions were constructed by joining the mouse ONS-M21 VL region and the first humanized version within CDR2. The hybrid VL regions revealed that residues in FR1 and/or FR2 were critical in creating a functional antigen binding site. Redesigning several versions with alterations in FR1 and FR2 revealed that the Pro-46 residue was the only critical residue for creating an antigen binding site. This approach should be helpful in identifying key residues in difficult cases of antibody humanization.

Usefulness of a Mouse Myelin Basic Protein Promoter for Gene Therapy of Malignant Glioma: Myelin Basic Protein Promoter Is Strongly Active in Human Malignant Glioma Cells

We have searched for suitable promoters to regulate the expression of suicide genes for use in gene therapy. We have shown that the 1.3‐kb fragment of the mouse myelin basic protein (MBP) promoter region initiates transcription in mouse glioma cells more efficiently than glial flbrillary acidic protein (GFAP) or myelin proteolipid protein (PLP) promoter. Among three different lengths of the MBP promoter, the shortest (256‐bp) core promoter region initiates transcription as efficiently as 650‐bp or 1.3‐kh MBP promoter lengths in RSV‐M glioma cells. To assess the suitability of the MBP promoter for use in clinical trials of malignant glioma gene therapy, we also had to show that it (the 1.3‐kb length in this case) Is effective in human glioma cells, as well as in murine glioma cells. The activity of the MBP promoter is much higher than that of GFAP or PLP promoter in most human glioma cells, suggesting that the MBP promoter would be best for directing toxic gene expression in gene therapy for patients with malignant glioma. Human glioma cells in which the MBP promoter was strongly active were sensitive to ganciclovir when they were transduced with MBP promoter/herpes simplex virus thymidine kinase gene‐bearing retroviruses. In conclusion, retrovirus‐targeted gene therapy for malignant glioma using this MBP promoter is a promising candidate for clinical trials.

Retrovirus-mediated gene transfer targeted to malignant glioma cells in murine brain

A murine model for meningeal metastasis of malignant glioma was developed to study selective gene transfer into tumor cells and to establish a reliable means of determining the rate of tumor cell infection. A murine ecotropic retroviral vector was created in which the Escherichia coliβ‐galactosidase gene served as a marker for gene expression from the integrated retrovirus. This retrovirus exhibited a high rate of infectivity in RSV‐M mouse glioma cells in vitro. The recombinant retrovirus was injected directly into the cisterna magna of the mice. Staining of β‐galactosidase showed that the rate of gene integration was high in the disseminated glioma cells. These results suggest the possibility of retrovirus‐mediated gene therapy for meningeal dissemination of malignant glioma.

Expression of Proteolipid Protein Gene Is Directly Associated with Secretion of a Factor Influencing Oligodendrocyte Development

Abstract: Oligodendroglial cell death in the myelin proteolipid protein (PLP) mutants can be partially rescued by the environment factor(s) supplied by the wild‐type cells in vivo and in vitro. It is possible that the presence of PLP or DM‐20 results in secretion of a factor or factors in the CNS influencing oligodendrocyte development. We previously showed that DM‐20 mRNA is produced in G26 mouse oligodendroglioma, B104 rat neuroblastoma, and B16 mouse melanoma but not in NIH3T3 mouse fibroblast cell lines. Culture supernatants from these cell lines were added to primary glial cell cultures from embryonic day 17 mouse brain. After 4 days, the number of oligodendrocytes present in cultures with supernatants from DM‐20‐producing cells (G26, B104, and B16) was significantly higher than that of control cultures but not with the NIH3T3 supernatant. To investigate more directly whether the PLP gene expression is involved in this process, NIH3T3 cells (nonneural cells) were forced to produce PLP or DM‐20. By addition of the supernatants from the PLP/DM‐20 transformants, the number of oligodendrocytes in the mixed glial cell cultures increased. This clearly demonstrates that the expression of the PLP gene is sufficient for and directly associated with secretion of a factor, which influences the oligodendrocyte development.

Monoclonal antibody ONS-M21 recognizes integrin α3 in gliomas and medulloblastomas

SummaryThe monoclonal antibody ONS-M21 recognizes an antigen found on the surface of glioma and medulloblastoma cells but does not react with the antigens of normal brain tissue. We purified and identified the 140-kDa protein by means of an antibody-binding affinity column. This 140-kDa antigen has sequences homologous to the amino-terminal region and five parts of the internal domain of integrin α3. When the integrin α3-related sequences was amplified and used to analyse the mRNA of glioma and medulloblastoma surgical specimens, the transcription level of integrin α3 mRNA appeared to be quantitatively correlated with the grade of malignancy. These findings suggest that the ONS-M21 antibody, which reacts with integrin α3, might be useful in the diagnosis of gliomas and medulloblastomas.

Eradication of murine brain tumors by direct inoculation of concentrated high titer-recombinant retrovirus harboring the herpes simplex virus thymidine kinase gene

Implantation of retrovirus-producing cells within a tumor has been demonstrated to eliminate malignant brain tumors effectively in animal models. In our previous study, the implantation of high-titer retrovirus-producing fibroblasts into tumors resulted in highly efficient transduction in vivo. The transduced glioma cells migrated far from the implantation site, potentiating the induction of the remarkable bystander effect. It is also possible, however, that the implantation of murine fibroblast-derived virus-producing cells may induce an immune response in patients. In this study, we prepared retroviruses carrying the herpes simplex virus thymidine kinase (HTK) gene with titers of 1.4–2.5 × 1011 colony-forming units (c.f.u.)/ml, and stereotactically inoculated only 3 μl of the HTK-bearing retroviruses into the brain tumors of mice. Following repetitive ganciclovir (GCV) intraperitoneal injection, effective killing of glioma cells in the mouse brain was observed. The transduction efficiency was nearly as high as that observed for the implantation of high-titer retrovirus-producing fibroblasts. Eighty percent of brain tumor-bearing mice were completely cured by our treatment protocol using concentrated HTK-harboring retroviruses. Our results suggest that repeated inoculations of high-titer retroviruses carrying the HTK gene followed by GCV treatment may be a promising strategy for the clinical treatment of malignant gliomas.

Gene delivery by HVJ-liposome in the experimental gene therapy of murine glioma.

We investigated the delivery of foreign genes into mouse glioma cells in vivo using hemagglutinating virus of Japan (HVJ)-liposomes, which are coated by Sendai virus envelope protein. HVJ-liposomes, containing lacZ gene or herpes simplex virus thymidine kinase (HSVtk) gene-bearing plasmid DNA were applied in the meningeal gliomatosis (MG) mouse model system. Highly efficient delivery was observed in disseminated glioma cells, and 80% of MG mice expressing the HSVtk gene were cured by treatment with ganciclovir. These results suggest that this novel gene delivery system may be applicable for the in vivo gene therapy of human malignant glioma.

Systemic interleukin 12 displays anti-tumour activity in the mouse central nervous system

In various systemic cancers, interleukin 12 (IL-12) induces anti-tumour immunity mediated by T lymphocytes and natural killer cells. To determine whether IL-12 has anti-tumour activity against malignant gliomas in the central nervous system (CNS), which is considered to be an immunologically privileged site, we treated mice with meningeal gliomatosis by intraperitoneal (i.p.) or intrathecal (i.t.) administration of recombinant murine IL-12. Although untreated mice revealed symptoms, such as body weight loss or paraplegia as a result of the meningeal gliomatosis within 8 days after tumour inoculation, 80% of the mice treated with IL-12 at 0.5 microg i.p. were cured. Many lymphocytes, mostly CD4+ and CD8+ cells, infiltrated to the tumours of IL-12-treated mice. The numbers of these cells increased in the cervical lymph nodes, into which the cerebrospinal fluid drains, and there they secreted a considerable amount of interferon-gamma. Mice cured by IL-12 rejected subcutaneous or i.t. rechallenge with their original glioma cells, but the same mice were not able to reject other syngeneic tumour cells. These results indicate that the immune system recognizes malignant glioma cells in the subarachnoid space of the CNS and that systemic IL-12 may produce effective anti-tumour activity and long-lasting tumour-specific immunity.

Transduction of glioma cells using a high-titer retroviral vector system and their subsequent migration in brain tumors.

The intracranial migration of transduced glioma cells was investigated in order to improve the treatment of malignant glioma by gene therapy using retroviral vectors. In this study, about half the volume of the tumor mass could be transduced in 14 days after only a single implantation of 3 × 105 retrovirus-producing cells into a tumor mass with a diameter of 5 mm. Moreover, we were able to follow the migration of glioma cells transduced by the lacZ-harboring retroviruses originating from the high-titer retrovirus-producing cells. Besides the importance of using a high-titer retroviral vector system, our results also indicate that the implantation site of the virus-producing cells and the interval between the implantation of the virus-producing cells and the subsequent administration of ganciclovir are important factors for the efficient killing of glioma cells.