Shigeo Koido

Fusions of Human Ovarian Carcinoma Cells with Autologous or Allogeneic Dendritic Cells Induce Antitumor Immunity

Human ovarian carcinomas express the CA-125, HER2/neu, and MUC1 tumor-associated Ags as potential targets for the induction of active specific immunotherapy. In the present studies, human ovarian cancer cells were fused to human dendritic cells (DC) as an alternative strategy to induce immunity against known and unidentified tumor Ags. Fusions of ovarian cancer cells to autologous DC resulted in the formation of heterokaryons that express the CA-125 Ag and DC-derived costimulatory and adhesion molecules. Similar findings were obtained with ovarian cancer cells fused to allogeneic DC. The fusion cells were functional in stimulating the proliferation of autologous T cells. The results also demonstrate that fusions of ovarian cancer cells to autologous or allogeneic DC induce cytolytic T cell activity and lysis of autologous tumor cells by a MHC class I-restricted mechanism. These findings demonstrate that fusions of ovarian carcinoma cells and DC activate T cell responses against autologous tumor and that the fusions are functional when generated with either autologous or allogeneic DC.

Induction of Antitumor Immunity by Vaccination of Dendritic Cells Transfected with MUC1 RNA

Dendritic cells (DC) are potent APCs. In this study, murine bone marrow-derived DC were transfected with RNA encoding the MUC1 Ag that is aberrantly overexpressed in human breast and other carcinomas. The MUC1 RNA-transfected DC exhibited cell surface expression of MUC1 and costimulatory molecules. After injection at the base of the tail, the transfected DC were detectable in inguinal lymph nodes by dual immunochemical staining. Vaccination of wild-type mice with MUC1 RNA-transfected DC induced anti-MUC1 immune responses against MUC1-positive MC38/MUC1, but not MUC1-negative, tumor cells. Mice immunized with the transfected DC were protected against challenge with MC38/MUC1 tumor cells. Furthermore, mice with established MC38/MUC1 tumors were eliminated after receiving the vaccination. CTLs isolated from mice immunized with the transfected DC exhibited specific cytolytic activity against MC38/MUC1 tumor cells. In contrast to these findings, there was little if any anti-MUC1 immunity induced with the transfected DC in MUC1 transgenic (MUC1.Tg) mice. However, coadministration of the transfected DC and IL-12 reversed the unresponsiveness to MUC1 Ag in MUC1.Tg mice and induced MUC1-specific immune responses. These findings demonstrate that vaccination of DC transfected with MUC1 RNA and IL-12 reverses tolerance to MUC1 and induces immunity against MUC1-positive tumors.

Prevention of Spontaneous Breast Carcinoma by Prophylactic Vaccination with Dendritic/Tumor Fusion Cells

Genetically modified mice with spontaneous development of mammary carcinoma provide a powerful tool to study the efficacy of tumor vaccines, since they mimic breast cancer development in humans. We used a transgenic murine model expressing polyomavirus middle T oncogene and mucin 1 tumor-associated Ag to determine the preventive effect of a dendritic/tumor fusion cell vaccine. The MMT (a transgenic murine model) mice developed mammary carcinoma between the ages of 65–108 days with 100% penetrance. No spontaneous CTL were detected. However, prophylactic vaccination of MMT mice with dendritic/tumor fusion cells induced polyclonal CTL activity against spontaneous mammary carcinoma cells and rendered 57–61% of the mice free of the disease at the end of experiment (180 days). Furthermore, the level of CTL activity was maintained with multiple vaccinations. The antitumor immunity induced by vaccination with dendritic/tumor fusion cells reacted differently to injected tumor cells and autochthonous tumor. Whereas the injected tumor cells were rejected, the autochthonous tumor evaded the attack and was allowed to grow. Collectively these results indicate that prophylactic vaccination with dendritic/tumor fusion cells confers sufficient antitumor immunity to counter the tumorigenesis of potent oncogenic products. The findings in the present study are highly relevant to cancers in humans.

The kinetics of in vivo priming of CD4 and CD8 T cells by dendritic/tumor fusion cells in MUC1-transgenic mice.

Previous work has demonstrated that dendritic/tumor fusion cells induce potent antitumor immune responses in vivo and in vitro. However, little is known about the migration and homing of fusion cells after s.c. injection or the kinetics of CD4+ and CD8+ T cell activation. In the present study, fluorescence-labeled dendritic/MUC1-positive tumor fusion cells (FC/MUC1) were injected s.c. into MUC1-transgenic mice. The FC/MUC1 migrated to draining lymph nodes and were closely associated with T cells in a pattern comparable with that of unfused dendritic cells. Immunization of MUC1-transgenic mice with FC/MUC1 resulted in proliferation of T cells and induced MUC1-specific CD8+ CTL. Moreover, CD4+ T cells activated by FC/MUC1 were multifunctional effectors that produced IL-2, IFN-γ, IL-4, and IL-10. These findings indicate that both CD4+ and CD8+ T cells can be primed in vivo by FC/MUC1 immunization.

Immunotherapy of spontaneous mammary carcinoma with fusions of dendritic cells and mucin 1‐positive carcinoma cells

The tumour‐associated antigen mucin 1 (MUC1) is a multifunctional protein involved in protection of mucous membranes, signal transduction, and modulation of the immune system. More than 70% of cancers overexpress MUC1, making MUC1 a potential target for immunotherapy. In the present study, MUC1 transgenic mice were crossed with syngeneic strains that express the polyomavirus middle‐T oncogene (PyMT) driven by the mouse mammary tumour virus promoter long‐terminal repeat (MMTV‐LTR). The resultant breed (MMT mice) developed spontaneous MUC1‐expressing mammary carcinomas with 100% penetrance at 8–15u2003weeks of age. As found in human breast cancer, the mammary carcinoma in MMT mice arose in multiple stages. Immunization with fusions of dendritic cells and MUC1‐positive tumour cells (FC/MUC1) induced MUC1‐specific immune responses that blocked or delayed the development of spontaneous breast carcinomas. In contrast, there was no delay of tumour development in MMT mice immunized with irradiated MC38/MUC1 tumour cells. The efficacy of fusion cells was closely correlated with the timing of initial immunization. Immunization with FC/MUC1 initiated in MMT mice at <u200a1, 1–2 and 2–3u2003months of age rendered 33, 5 and 0% of mice free of tumour, respectively, up to 6u2003months. Whereas mice immunized in the later stage of tumour development succumbed to their disease, immunization resulted in control of tumour progression and prolongation of life. These results indicate that immunization with FC/MUC1 can generate an anti‐MUC1 response that is sufficient to delay the development of spontaneous mammary carcinomas and control tumour progression in MMT mice.

Selection and characterization of MUC1-specific CD8+ T cells from MUC1 transgenic mice immunized with dendritic-carcinoma fusion cells.

Mice transgenic for the human MUC1 carcinoma‐associated antigen (MUC1.Tg) are tolerant to immunization with MUC1 antigen. Recent studies, however, have demonstrated that immunization of MUC1.Tg mice with fusions of MUC1‐positive tumour and dendritic cells (FC/MUC1) reverses MUC1 unresponsiveness and results in rejection of established MUC1‐positive pulmonary metastases. Here we demonstrate that lymph node cells from MUC1.Tg mice immunized with the FC/MUC1 fusion cells proliferate in response to MUC1 antigen by a mechanism dependent on the function of CD4, major histocompatibility complex (MHC) class II, B7‐1, B7‐2, CD28, CD40 and CD40 ligand. The findings demonstrate that stimulation of lymph node cells with MUC1 results in selection of MUC1‐specific CD8+ T cells. We show that the CD8+ T cells exhibit MUC1‐specific cytotoxic T lymphocyte (CTL) activity by recognition of MUC1 peptides presented in the context of MHC class I molecules Kb and Db. The MUC1‐specific CD8+ T cells also exhibit antitumour activity against MUC1‐positive metastases, but with no apparent reactivity against normal tissues. These results indicate that immunization of MUC1.Tg mice with FC/MUC1 reverses immunological unresponsiveness to MUC1 by presentation of MUC1 peptides in the presence of costimulatory signals and generates MHC‐restricted MUC1‐specific CD8+ T cells.

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.

Treatment with Chemotherapy and Dendritic Cells Pulsed with Multiple Wilms' Tumor 1 (WT1)–Specific MHC Class I/II–Restricted Epitopes for Pancreatic Cancer

Purpose: We performed a phase I trial to investigate the safety, clinical responses, and Wilms tumor 1 (WT1)-specific immune responses following treatment with dendritic cells (DC) pulsed with a mixture of three types of WT1 peptides, including both MHC class I and II–restricted epitopes, in combination with chemotherapy. Experimental Design: Ten stage IV patients with pancreatic ductal adenocarcinoma (PDA) and 1 patient with intrahepatic cholangiocarcinoma (ICC) who were HLA-positive for A*02:01, A*02:06, A*24:02, DRB1*04:05, DRB1*08:03, DRB1*15:01, DRB1*15:02, DPB1*05:01, or DPB1*09:01 were enrolled. The patients received one course of gemcitabine followed by biweekly intradermal vaccinations with mature DCs pulsed with MHC class I (DC/WT1-I; 2 PDA and 1 ICC), II (DC/WT1-II; 1 PDA), or I/II–restricted WT1 peptides (DC/WT1-I/II; 7 PDA), and gemcitabine. Results: The combination therapy was well tolerated. WT1-specific IFNγ-producing CD4+ T cells were significantly increased following treatment with DC/WT1-I/II. WT1 peptide-specific delayed-type hypersensitivity (DTH) was detected in 4 of the 7 patients with PDA vaccinated with DC/WT1-I/II and in 0 of the 3 patients with PDA vaccinated with DC/WT1-I or DC/WT1-II. The WT1-specific DTH-positive patients showed significantly improved overall survival (OS) and progression-free survival (PFS) compared with the negative control patients. In particular, all 3 patients with PDA with strong DTH reactions had a median OS of 717 days. Conclusions: The activation of WT1-specific immune responses by DC/WT1-I/II combined with chemotherapy may be associated with disease stability in advanced pancreatic cancer. Clin Cancer Res; 20(16); 4228–39. ©2014 AACR.

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.