Sadamu Homma

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.

Clinical and immunologic evaluation of dendritic cell-based immunotherapy in combination with gemcitabine and/or S-1 in patients with advanced pancreatic carcinoma

Objectives In the current study, we have evaluated the clinical and immunological responses in patients with advanced pancreatic carcinoma who received dendritic cell (DC)–based immunotherapy in combination with gemcitabine and/or S-1. Methods Dendritic cell–based immunotherapy (DC vaccine alone or DC vaccine plus lymphokine-activated killer [LAK] cell therapy) in combination with gemcitabine and/or S-1 has been carried out in 49 patients with inoperable pancreatic carcinoma refractory to standard treatment. Results Of 49 patients, 2 patients had complete remission, 5 had partial remission, and 10 had stable disease. Prolongation of survival in this cohort was highly likely (median survival, 360 days). Survival of patients receiving DC vaccine and chemotherapy plus LAK cell therapy was longer than those receiving DC vaccine in combination with chemotherapy but no LAK cells. Increased numbers of cancer antigen-specific cytotoxic T cells and decreased regulatory T cells were observed in several patients on immunotherapy, but increased overall survival time tended to be associated only with the latter. None of the patients experienced grade 3 or worse adverse events during the treatment period. Conclusions Dendritic cell vaccine–based immunotherapy combined with chemotherapy was shown to be safe and possibly effective in patients with advanced pancreatic cancer refractory to standard treatment.

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.

Current Immunotherapeutic Approaches in Pancreatic Cancer

Pancreatic cancer is a highly aggressive and notoriously difficult to treat. As the vast majority of patients are diagnosed at advanced stage of the disease, only a small population is curative by surgical resection. Although gemcitabine-based chemotherapy is typically offered as standard of care, most patients do not survive longer than 6 months. Thus, new therapeutic approaches are needed. Pancreatic cancer cells that develop gemcitabine resistance would still be suitable targets for immunotherapy. Therefore, one promising treatment approach may be immunotherapy that is designed to target pancreatic-cancer-associated antigens. In this paper, we detail recent work in immunotherapy and the advances in concept of combination therapy of immunotherapy and chemotherapy. We offer our perspective on how to increase the clinical efficacy of immunotherapies for pancreatic cancer.

Immunotherapy for colorectal cancer

The incidence of colorectal cancer (CRC) is on the rise, and the prognosis for patients with recurrent or metastatic disease is extremely poor. Although chemotherapy and radiation therapy can improve survival rates, it is imperative to integrate alternative strategies such as immunotherapy to improve outcomes for patients with advanced CRC. In this review, we will discuss the effect of immunotherapy for inducing cytotoxic T lymphocytes and the major immunotherapeutic approaches for CRC that are currently in clinical trials, including peptide vaccines, dendritic cell-based cancer vaccines, whole tumor cell vaccines, viral vector-based cancer vaccines, adoptive cell transfer therapy, antibody-based cancer immunotherapy, and cytokine therapy. The possibility of combination therapies will also be discussed along with the challenges presented by tumor escape mechanisms.

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.

Wilms tumor gene (WT1) peptide-based cancer vaccine combined with gemcitabine for patients with advanced pancreatic cancer.

Wilms tumor gene (WT1) protein is an attractive target for cancer immunotherapy. We aimed to investigate the feasibility of a combination therapy consisting of gemcitabine and WT1 peptide–based vaccine for patients with advanced pancreatic cancer and to make initial assessments of its clinical efficacy and immunologic response. Thirty-two HLA-A*24:02+ patients with advanced pancreatic cancer were enrolled. Patients received HLA-A*24:02-restricted, modified 9-mer WT1 peptide (3 mg/body) emulsified with Montanide ISA51 adjuvant (WT1 vaccine) intradermally biweekly and gemcitabine (1000 mg/m2) on days 1, 8, and 15 of a 28-day cycle. This combination therapy was well tolerated. The frequencies of grade 3–4 adverse events for this combination therapy were similar to those for gemcitabine alone. Objective response rate was 20.0% (6/30 evaluable patients). Median survival time and 1-year survival rate were 8.1 months and 29%, respectively. The association between longer survival and positive delayed-type hypersensitivity to WT1 peptide was statistically significant, and longer survivors featured a higher frequency of memory-phenotype WT1-specific cytotoxic T lymphocytes both before and after treatment. WT1 vaccine in combination with gemcitabine was well tolerated for patients with advanced pancreatic cancer. Delayed-type hypersensitivity-positivity to WT1 peptide and a higher frequency of memory-phenotype WT1-specific cytotoxic T lymphocytes could be useful prognostic markers for survival in the combination therapy with gemcitabine and WT1 vaccine. Further clinical investigation is warranted to determine the effectiveness of this combination therapy.

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.

Gemcitabine enhances Wilms’ tumor gene WT1 expression and sensitizes human pancreatic cancer cells with WT1-specific T-cell-mediated antitumor immune response

Wilms’ tumor gene (WT1), which is expressed in human pancreatic cancer (PC), is a unique tumor antigen recognized by T-cell-mediated antitumor immune response. Gemcitabine (GEM), a standard therapeutic drug for PC, was examined for the regulation of WT1 expression and the sensitizing effect on PC cells with WT1-specific antitumor immune response. Expression of WT1 was examined by quantitative PCR, immunoblot analysis, and confocal microscopy. Antigenic peptide of WT1 presented on HLA class I molecules was detected by mass spectrometry. WT1-specific T-cell receptor gene–transduced human T cells were used as effecter T cells for the analysis of cytotoxic activity. GEM treatment of human MIAPaCa2 PC cells enhanced WT1 mRNA levels, and this increase is associated with nuclear factor kappa B activation. Tumor tissue from GEM-treated MIAPaCa2-bearing SCID mice also showed an increase in WT1 mRNA. Some human PC cell lines other than MIAPaCa2 showed up-regulation of WT1 mRNA levels following GEM treatment. GEM treatment shifted WT1 protein from the nucleus to the cytoplasm, which may promote proteasomal processing of WT1 protein and generation of antigenic peptide. In fact, presentation of HLA-A*2402-restricted antigenic peptide of WT1 (CMTWNQMNL) increased in GEM-treated MIAPaCa2 cells relative to untreated cells. WT1-specific cytotoxic T cells killed MIAPaCa2 cells treated with an optimal dose of GEM more efficiently than untreated MIAPaCa2 cells. GEM enhanced WT1 expression in human PC cells and sensitized PC cells with WT1-specific T-cell-mediated antitumor immune response.

Streptococcal Preparation OK-432 Promotes Fusion Efficiency and Enhances Induction of Antigen-Specific CTL by Fusions of Dendritic Cells and Colorectal Cancer Cells

Dendritic/tumor fusion cell (FC) vaccine is an effective approach for various types of cancer but has not yet been standardized. Antitumor activity can be modulated by different mechanisms such as dendritic cell (DC) maturation state. This study addressed optimal strategies for FC preparations to enhance Ag-specific CTL activity. We have created three types of FC preparations by alternating fusion cell partners: 1) immature DCs fused with autologous colorectal carcinoma cells (Imm-FCs); 2) Imm-FCs followed by stimulation with penicillin-inactivated Streptococcus pyogenes (OK-432) (Imm-FCs/OK); and 3) OK-432-stimulated DCs directly fused to autologous colorectal carcinoma cells (OK-FCs). Both OK-FCs and Imm-FCs/OK coexpressed the CEA, MUC1, and significantly higher levels of CD86, CD83, and IL-12 than those obtained with Imm-FCs. Short-term culture of fusion cell preparations promoted the fusion efficiency. Interestingly, OK-FCs were more efficient in stimulating CD4+ and CD8+ T cells capable of high levels of IFN-γ production and cytolysis of autologous tumor or semiallogeneic targets. Moreover, OK-FCs are more effective inducer of CTL activation compared with Imm-FCs/OK on a per fusion cell basis. The pentameric assay confirmed that CEA- and MUC1-specific CTL was induced simultaneously by OK-FCs at high frequency. Furthermore, the cryopreserved OK-FCs retained stimulatory capacity for inducing antitumor immunity. These results suggest that OK-432 promotes fusion efficiency and induction of Ag-specific CTL by fusion cells. We conclude that DCs fused after stimulation by OK-432 may have the potential applicability to the field of antitumor immunotherapy and may provide a platform for adoptive immunotherapy in the clinical setting.