Tsuneya Ohno

Vaccination of Glioma Patients with Fusions of Dendritic and Glioma Cells and Recombinant Human Interleukin 12

Despite aggressive treatment, the median survival of patients with high-grade malignant astrocytoma is about 1 year. The authors investigated the safety and clinical response to immunotherapy using fusions of dendritic and glioma cells combined with recombinant human interleukin 12 (rhIL-12) for the treatment of malignant glioma. Fifteen patients with malignant glioma participated in this study. Dendritic cells were generated from peripheral blood. Cultured autologous glioma cells were established from surgical specimens in each case. Fusion cells were prepared from dendritic and glioma cells using polyethylene glycol. All patients received fusion cells intradermally on day 1. rhIL-12 was injected subcutaneously at the same site on days 3 and 7. Response to the treatment was evaluated by clinical observations and radiologic findings. No serious adverse effects were observed. In four patients, magnetic resonance imaging showed a greater than 50% reduction in tumor size. One patient had a mixed response. These results show that administration of fusion cells and rhIL-12 safely induces clinical antitumor effects in some patients with malignant glioma.

Use of a murine monoclonal antibody for detection of circulating plasma DF3 antigen levels in breast cancer patients.

The murine monoclonal antibody (MAb), designated DF3, reacts with a 300,000-mol wt mammary epithelial antigen. A sequential double-determinant radioimmunoassay (RIA) has been developed to monitor circulating DF3 antigen. Using this assay, we have demonstrated that 33 of 36 normal women had plasma RIA antigen levels less than 150 U/ml. In contrast, 33 of 43 patients (76%) with metastatic breast cancer had RIA DF3 antigen levels greater than or equal to 150 U/ml. The difference between these two groups was statistically significant (P less than 0.001). Similar results have been obtained with a double-determinant enzyme-linked immunoassay (EIA). Only 6 of 111 age-matched normal subjects had EIA DF3 antigens levels greater than or equal to 30 U/ml, while 42 of 58 patients (72%) with breast cancer had levels equal to or above this value. Thus, similar patterns of specificity are obtained with the EIA or RIA. The elevation of circulating DF3 antigen levels in breast cancer patients has been confirmed by transfer blot assays. MAb DF3 reactivity occurred predominantly with circulating antigens of three different molecular weights ranging from 300,000 to approximately 400,000 mol wt. We also demonstrate that patients with both primary and metastatic breast cancer who were free of detectable disease at the time of sampling have DF3 antigen levels that are similar to those obtained from normal subjects. While patients with hepatoma (27%) and ovarian carcinoma (47%) also had elevated circulating DF3 antigen levels, the results suggest that DF3 antigen levels may be useful in distinguishing breast cancer patients from those with esophageal, gastric, colorectal, pancreatic, and lung carcinomas. Furthermore, the results of the RIA, EIA, and transblot analyses demonstrate that the measurement of circulating DF3 antigen levels provides a new and potentially useful marker to follow the clinical course of patients with metastatic breast cancer.

Antitumor effect of immunizations with fusions of dendritic and glioma cells in a mouse brain tumor model.

The authors studied antitumor immunity conferred by fusions of dendritic and glioma cells in a mouse brain tumor model. Previous immunization with fusion cells (FCs) prevented tumor formation on challenge with glioma cells in the flank or in the brain. Efficacy was decreased when studies were performed in mice depleted of CD8+ cells. In a treatment model, FCs were injected subcutaneously after tumor development in the brain. The administration of FCs alone had limited effects on survival of mice bearing brain tumors. Importantly, however, administration of FCs and recombinant interleukin-12 (rIL-12) remarkably prolonged the survival of mice with brain tumors. Cytotoxic T lymphocyte activity against glioma cells from immunized mice was also stimulated by coadministration of FCs and rIL-12 compared with that obtained with FCs or rIL-12 alone. These data support the therapeutic efficacy of combining FC-based vaccine therapy and rIL-12.

Telomerase overexpression in K562 leukemia cells protects against apoptosis by serum deprivation and double-stranded DNA break inducing agents, but not against DNA synthesis inhibitors

Telomeres are specialized DNA/protein structures that act as protective caps to prevent end fusions. The maintenance of telomeres is essential for chromosomal stability. Telomerase is regulated by human telomerase reverse transcriptase (hTERT). c-Myc oncoprotein is also implicated in the positive regulation of hTERT expression. We show here that two clones of hTERT-transfected K562 erythroleukemia cells have elongated telomeres (22.5 and 24.0 kb), whereas telomere length of both c-Myc-transfected K562 cells and parental K562 cells is 6.5 kb. Telomerase activity and hTERT mRNA expression increased in hTERT-transfected K562 cells, while the expression levels of telomerase activity and hTERT in c-Myc-transfected K562 cells were similar to that in parental K562 cells, despite an overexpression of c-Myc. Importantly, we found that hTERT-transfected K562 cells are protected against apoptosis induced by serum deprivation and double-stranded DNA break inducing agents (ionizing irradiation, and etoposide (VP-16)), but not against DNA synthesis inhibitors (1-beta-D-arabinofuranosylcytosine and hydroxyurea). These findings suggest that overexpression of telomerase by transfecting hTERT confers telomere-elongation and resistance to double-stranded DNA break inducing agents.

Preventive antitumor activity against hepatocellular carcinoma (HCC) induced by immunization with fusions of dendritic cells and HCC cells in mice.

Background. The prevention of recurrence of hepatocellular carcinoma (HCC) after treatment is very important for improvement of the prognosis of HCC patients. Dendritic cells (DCs) are potent antigen-presenting cells that can prime naive T cells to induce a primary immune response. We attempted to induce preventive antitumor immunity against HCC by immunizing BALB/c mice with fusions of DCs and HCC cells. Methods. Murine bone marrow-derived DCs and a murine HCC cell line, BNL cells, were fused by treatment with 50% polyethyleneglycol (PEG). Fusion efficacy was assessed by the analysis of fusions of BNL cells stained with red fluorescent dye and DCs stained with green fluorescent dye. Mice injected intravenously with DC/BNL fusions were challenged by BNL cell inoculation. Results. About 30% of the PEG-treated nonadherent cells with both fluorescences were considered to be fusion cells. The cell fraction of DC/BNL fusions showed phenotypes of DCs, MHC class II, CD80, CD86, and intercellular adhesion molecule (ICAM)-1, which were not expressed on BNL cells. Mice immunized with the fusions were protected against the inoculation of BNL tumor cells, whereas injection with a mixture of DCs and BNL cells not treated with PEG did not provide significant resistance against BNL cell inoculation. Splenocytes from DC/BNL fusion-immunized mice showed lytic activity against BNL cells. Conclusions. These results demonstrate that immunization with fusions of DCs and HCC cells is capable of inducing preventive antitumor immunity against HCC.

Antitumour activity mediated by CD4+ cytotoxic T lymphocytes against MHC class II‐negative mouse hepatocellular carcinoma induced by dendritic cell vaccine and interleukin‐12

When BALA/c mice with BNL hepatocellular carcinoma (HCC) were treated with dendritic cells fused with BNL cells (DC/BNL) and recombinant murine interleukin (IL)‐12, tumour development was significantly suppressed, whereas treatment with either DC/BNL or IL‐12 alone did not show a tumour‐suppressive effect. Antitumour activity induced by DC/BNL + IL‐12 was abrogated by depletion of CD4+ T cells, but not by depletion of CD8+ T cells or natural killer cells. Splenic CD4+ T cells and CD8+ T cells from DC/BNL‐treated mice showed cytotoxic activity against BNL cells after 3 days of incubation with DC/BNL, although BNL cells do not express major histocompatibility complex (MHC) class II molecules even after treatment with interferon (INF)‐γ. Furthermore, CD4+ T cells killed syngeneic‐irrelevant CT26 cells and even allogeneic Hepa1‐6 cells. This cytotoxicity was blocked by concanamycin A, but not by an anti‐Fas ligand (FasL) monoclonal antibody, indicating that cytotoxic activity was mediated by perforin. Immunofluorescence microscopy demonstrated that abundant CD4+ T cells and MHC class II‐positive macrophages, but not CD8+ T cells, had infiltrated tumour tissue in mice treated with DC/BNL + IL‐12. Flow cytometric analysis of tumour‐infiltrating cells in mice treated with DC/BNL + IL‐12 showed increases in CD4+ T cells and MHC class II+ CD11b+ cells but not in CD8+ T cells or MHC class I+ CD11b+ cells. Our results suggest that, in BNL‐bearing mice treated with DC/BNL + IL‐12, tumour macrophages activated by INF‐γ produced by IL‐12‐stimulated T cells might present BNL tumour antigens and activate DC/BNL‐primed CD4+ cytotoxic T lymphocytes (CTLs) in a MHC class II‐dependent manner, leading to perforin‐mediated bystander killing of neighbouring MHC class II‐negative tumour cells.

Prevention of gastrointestinal tumors based on adenomatous polyposis coli gene mutation by dendritic cell vaccine

Here we describe the effect of immunization with dendritic cells loaded with syngeneic tumor cells (DC/Ts) by polyethylene glycol treatment, on tumor development in adenomatous polyposis coli (APC) gene mutant mouse models, APC1309 and APC(Min-/+), in which adenomatous polyps of the gastrointestinal tracts develop with a high incidence. Treatment with DC/Ts prevented the development of gastrointestinal tumors, and coadministration of DC/Ts and IL-12 caused a further reduction in tumor incidence. Splenocytes from APC1309 mice treated with DC/Ts and IL-12 showed no cytotoxic activity toward the tumor cells, but serum antibody specific to them was detected. IgG from the treated mice exhibited cytotoxic activity against the tumor cells in vitro. Predominance of Th2 cell response over Th1 response was also suggested by ELISPOT assays in the treated mice. Depletion in vivo of CD4(+) T cells, not CD8(+) T cells, by the intraperitoneal administration of corresponding mAbs decreased the antitumor effect of DC/T inoculation. Immunofluorescence microscopic studies showed that Ig was attached to tumor cells in mice treated with DC/Ts and IL-12. These findings indicate that DC/T vaccination prevents tumor development through APC gene mutation and that its preventive effects are mediated by humoral antitumor immunity.

Induction of effective antitumor immunity in a mouse brain tumor model using B7‐1 (CD80) and intercellular adhesive molecule 1 (ICAM‐1; CD54) transfection and recombinant interleukin 12

Although tumor‐specific T lymphocytes recognize tumor‐associated antigens (TAA) present on their cell surface via major histocompatibility complex (MHC) molecules, T cells require other activating signals. These are provided by costimulatory molecules, including B7‐1 (CD80), B7‐2 (CD86) and intercellular adhesive molecule 1 (ICAM‐1; CD54). Transfecting mouse tumor cell lines with the B7 gene can lead to primary tumor rejection and the establishment of protective immunity. However, some studies have shown that the B7 effect upon T‐cell‐dependent tumor immunity is limited. Therefore, we examined the antitumor effects of recombinant interleukin 12 (IL‐12) and genetically engineered glioma cells expressing B7‐1 or both B7‐1 and ICAM‐1. Vaccination of mice with B7‐1‐expressing tumor cells substantially inhibited the growth of subcutaneously inoculated gliomas but not those located in the brain. Vaccination with B7‐1‐expressing tumor cells and systemic recombinant IL‐12 (rIL‐12) was more effective than either B7‐1‐expressing tumor cells or rIL‐12 alone. Our murine brain tumor model also showed that vaccination with tumor cells expressing both B7‐1 and ICAM‐1 combined with rIL‐12 prolonged survival. We have demonstrated the therapeutic potential of vaccination with rIL‐12 and tumor cells expressing both B7‐1 and ICAM‐1 in the control of glioma growth. Int. J. Cancer 82:714–720, 1999.

Cancer immunotherapy using dendritic/tumour-fusion vaccine induces elevation of serum anti-nuclear antibody with better clinical responses

Dendritic cell (DC) vaccines might induce both anti‐tumour immunity and autoimmunity. In this report, we demonstrate elevated levels of anti‐nuclear antibody (ANA) in the sera of patients with cancer who had received immunotherapy with a dendritic/tumour‐fusion vaccine. Twenty‐two patients were treated with DC vaccine of fusion cells composed of autologous DCs and tumour cells (DC/tumour‐fusion vaccine), which was generated by treating each cell type with polyethylene glycol. Nine of the 22 patients were treated with both the DC/tumour‐fusion vaccine and systemic administration of recombinant human interleukin (rhIL)‐12. Serum levels of ANA were examined with an enzyme‐linked immunosorbent assay kit. One patient with gastric carcinoma (patient 1, DC/tumour‐fusion vaccine alone), one patient with breast cancer (patient 2, DC/tumour‐fusion vaccine alone) and one patient with ovarian cancer (patient 3, DC/tumour‐fusion vaccine + rhIL‐12) showed significant elevations of serum ANA levels during treatment. In patient 1 malignant ascitic effusion resolved and serum levels of tumour markers decreased. Patients 2 and 3 remained in good physical condition during treatment for 24 and 9 months, respectively. Immunoblot analysis indicated antibody responses to autologous tumour cells after vaccination in patient 2. None of the treated patients showed clinical symptoms suggesting autoimmune disease. Patients with elevated serum levels of ANA had significantly longer treatment periods than those without it. Elevated serum levels of ANA after DC/tumour‐fusion cell vaccine might be associated with anti‐tumour immune response induced by the vaccination.

Gene transduction for disseminated intraperitoneal tumor using cationic liposomes containing non-histone chromatin proteins: cationic liposomal gene therapy of carcinomatosa.

Cationic liposome-mediated gene therapy of cancer has been examined in a disseminated intraperitoneal tumor model. A combination of the strong CAG promoter–enhancer unit and high mobility group 1,2 (HMG-1,2) proteins has been used to improve transfection efficiency. The optimal deoxyribonucleic acid (DNA) and HMG-1,2 concentration ratio was determined in in vitro studies. Subsequently, we administered the liposome–DNA–HMG-1,2 complex intraperitoneally to tumor bearing nude mice and defined the most efficient concentration of liposomes. Using this approach, the median survival of tumor bearing nude mice was prolonged by the administration of a human tumor necrosis factor-α (TNF-α) gene inserted into a eukaryotic strong expression vector (pcagTNF-α) and exogenously added interferon-γ (INF-γ) (72 ± 3 days: mean ± s.e.). By contrast, survival was 36 ± 3, 39 ± 3 and 46 ± 4 days in groups receiving the TNF-α gene inserted in the reverse orientation (pcagTNF-αR) and normal saline (NS), pcagTNF-αR and INF-γ, and pcagTNF-α and NS, respectively. These results demonstrate an efficient approach for gene therapy of disseminated intraperitoneal cancer.