Phase I/II trial of combination of temozolomide chemotherapy and immunotherapy with fusions of dendritic and glioma cells in patients with glioblastoma.

BACKGROUND: This trial was designed to evaluate the safety and clinical responses to a combination of temozolomide (TMZ) chemotherapy and immunotherapy with fusions of DCs and glioma cells in patients with glioblastoma (GBM). METHOD: GBM patients were assigned to two groups: a group of recurrent GBMs after failing TMZ-chemotherapy against the initially diagnosed glioma (Group-R) or a group of newly diagnosed GBMs (Group-N). Autologous cultured glioma cells obtained from surgical specimens were fused with autologous DCs using polyethylene glycol. The fusion cells (FC) were inoculated intradermally in the cervical region. Toxicity, progression-free survival (PFS), and overall survival (OS) of this trial were evaluated. Expressions of WT-1, gp-100, and MAGE-A3, recognized as chemoresistance-associated peptides (CAP), were confirmed by immunohistochemistry of paraffin-embedded tumor samples. Patient's PBMCs of pre- and post-vaccination were evaluated by tetramer and ELISPOT assays. RESULTS: FC-immunotherapy was well tolerated in all patients. Medians of PFS and OS of Group-R (n = 10) were 10.3 and 18.0 months, and those of Group-N (n = 22) were 18.3 and 30.5 months, respectively. Up-regulation and/or cytoplasmic accumulation of CAPs was observed in the recurrent tumors of Group-R patients compared with their initially excised tumors. Specific immune responses against CAPs were observed in the tetramer and ELISPOT assays. CONCLUSIONS: The combination of TMZ-treatment leading to up-regulation and/or cytoplasmic accumulation of CAPs, with FC-immunotherapy as a means of producing specific immunity against CAPs, may safely induce anti-tumor effects in patients with GBM.

Up-regulation of HER2 by gemcitabine enhances the antitumor effect of combined gemcitabine and trastuzumab emtansine treatment on pancreatic ductal adenocarcinoma cells.

BACKGROUND: Although pancreatic ductal adenocarcinomas (PDAs) widely express HER2, the expression level is generally low. If HER2 expression in PDA cells could be enhanced by treatment with a given agent, then combination therapy with that agent and trastuzumab emtansine (T-DM1), a chemotherapeutic agent that is a conjugate of trastuzumab, might lead to significant antitumor effects against PDA. METHODS: Cell proliferation was examined by spectrophotometry. HER2 expression was examined by flow cytometry, immunoblot and quantitative reverse transcription polymerase chain reaction. T-DM1 binding to cells was examined by flow cytometry and enzyme-linked immunosorbent assay. RESULTS: Out of 5 tested human PDA cell lines, including MIA PaCa-2, three showed increases in HER2 expression after gemcitabine (GEM) treatment. The binding of T-DM1 to GEM-treated MIA PaCa-2 cells was higher than to untreated MIA PaCa-2 cells. Treatment with GEM and T-DM1 showed synergic cytotoxic effects on MIA PaCa-2 cells in vitro. Cells in the G2M phase of the cell cycle were retained after GEM treatment and showed higher levels of HER2 expression, possibly contributing to the synergic effect of GEM and T-DM1. CONCLUSIONS: Combined treatment with GEM and T-DM1 might confer a potent therapeutic modality against PDA as a result of GEM-mediated HER2 up-regulation.

Vaccination with vascular progenitor cells derived from induced pluripotent stem cells elicits antitumor immunity targeting vascular and tumor cells.

Vaccination of BALB/c mice with dendritic cells (DCs) loaded with the lysate of induced vascular progenitor (iVP) cells derived from murine-induced pluripotent stem (iPS) cells significantly suppressed the tumor of CMS-4 fibrosarcomas and prolonged the survival of CMS-4-inoculated mice. This prophylactic antitumor activity was more potent than that of immunization with DCs loaded with iPS cells or CMS-4 tumor cells. Tumors developed slowly in mice vaccinated with DCs loaded with iVP cells (DC/iVP) and exhibited a limited vascular bed. Immunohistochemistry and a tomato-lectin perfusion study demonstrated that the tumors that developed in the iVP-immunized mice showed a marked decrease in tumor vasculature. Immunization with DC/iVP induced a potent suppressive effect on vascular-rich CMS-4 tumors, a weaker effect on BNL tumors with moderate vasculature, and nearly no effect on C26 tumors with poor vasculature. Treatment of DC/iVP-immunized mice with a monoclonal antibody against CD4 or CD8, but not anti-asialo GM1, inhibited the antitumor activity. CD8(+) T cells from DC/iVP-vaccinated mice showed significant cytotoxic activity against murine endothelial cells and CMS-4 cells, whereas CD8(+) T cells from DC/iPS-vaccinated mice did not. DNA microarray analysis showed that the products of 29 vasculature-associated genes shared between genes upregulated by differentiation from iPS cells into iVP cells and genes shared by iVP cells and isolated Flk-1(+) vascular cells in CMS-4 tumor tissue might be possible targets in the immune response. These results suggest that iVP cells from iPS cells could be used as a cancer vaccine targeting tumor vascular cells and tumor cells.

Vaccines targeting tumor blood vessel antigens promote CD8(+) T cell-dependent tumor eradication or dormancy in HLA-A2 transgenic mice.

We have recently shown that effective cytokine gene therapy of solid tumors in HLA-A2 transgenic (HHD) mice lacking murine MHC class I molecule expression results in the generation of HLA-A2-restricted CD8(+) T effector cells selectively recognizing tumor blood vessel-associated pericytes and/or vascular endothelial cells. Using an HHD model in which HLA-A2(neg) tumor (MC38 colon carcinoma or B16 melanoma) cells are not recognized by the CD8(+) T cell repertoire, we now show that vaccines on the basis of tumor-associated blood vessel Ags (TBVA) elicit protective Tc1-dependent immunity capable of mediating tumor regression or extending overall survival. Vaccine efficacy was not observed if (HLA-A2(neg)) wild-type C57BL/6 mice were instead used as recipient animals. In the HHD model, effective vaccination resulted in profound infiltration of tumor lesions by CD8(+) (but not CD4(+)) T cells, in a coordinate reduction of CD31(+) blood vessels in the tumor microenvironment, and in the "spreading" of CD8(+) T cell responses to alternate TBVA that were not intrinsic to the vaccine. Protective Tc1-mediated immunity was durable and directly recognized pericytes and/or vascular endothelial cells flow-sorted from tumor tissue but not from tumor-uninvolved normal kidneys harvested from these same animals. Strikingly, the depletion of CD8(+), but not CD4(+), T cells at late time points after effective therapy frequently resulted in the recurrence of disease at the site of the regressed primary lesion. This suggests that the vaccine-induced anti-TBVA T cell repertoire can mediate the clinically preferred outcomes of either effectively eradicating tumors or policing a state of (occult) tumor dormancy.

Intratumoral IL-12 gene therapy results in the crosspriming of Tc1 cells reactive against tumor-associated stromal antigens.

HLA-A2 transgenic mice bearing established HLA-A2(neg) B16 melanomas were effectively treated by intratumoral (i.t.) injection of syngeneic dendritic cells (DCs) transduced to express high levels of interleukin (IL)-12, resulting in CD8(+) T cell-dependent antitumor protection. In this model, HLA-A2-restricted CD8(+) T cells do not directly recognize tumor cells and therapeutic benefit was associated with the crosspriming of HLA-A2-restricted type-1 CD8(+) T cells reactive against antigens expressed by stromal cells [i.e., pericytes and vascular endothelial cells (VEC)]. IL-12 gene therapy-induced CD8(+) T cells directly recognized HLA-A2(+) pericytes and VEC flow-sorted from B16 tumor lesions based on interferon (IFN)-γ secretion and translocation of the lytic granule-associated molecule CD107 to the T cell surface after coculture with these target cells. In contrast, these CD8(+) T effector cells failed to recognize pericytes/VEC isolated from the kidneys of tumor-bearing HHD mice. The tumor-associated stromal antigen (TASA)-derived peptides studied are evolutionarily conserved and could be recognized by CD8(+) T cells harvested from the blood of HLA-A2(+) normal donors or melanoma patients after in vitro stimulation. These TASA and their derivative peptides may prove useful in vaccine formulations against solid cancers, as well as, in the immune monitoring of HLA-A2(+) cancer patients receiving therapeutic interventions, such as IL-12 gene therapy.

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.

Immunologic monitoring of cellular responses by dendritic/tumor cell fusion vaccines.

Although dendritic cell (DC)- based cancer vaccines induce effective antitumor activities in murine models, only limited therapeutic results have been obtained in clinical trials. As cancer vaccines induce antitumor activities by eliciting or modifying immune responses in patients with cancer, the Response Evaluation Criteria in Solid Tumors (RECIST) and WHO criteria, designed to detect early effects of cytotoxic chemotherapy in solid tumors, may not provide a complete assessment of cancer vaccines. The problem may, in part, be resolved by carrying out immunologic cellular monitoring, which is one prerequisite for rational development of cancer vaccines. In this review, we will discuss immunologic monitoring of cellular responses for the evaluation of cancer vaccines including fusions of DC and whole tumor cell.

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.

Dendritic/pancreatic carcinoma fusions for clinical use: Comparative functional analysis of healthy- versus patient-derived fusions.

Fetal calf serum (FCS)-independent pancreatic cancer cells were established in plasma protein fraction (PPF)-supplemented medium that is an agent of good manufacturing practice (GMP) grade. Dendritic cells (DCs) were activated with the Toll-like receptor agonist, penicillin-inactivated Streptococcus pyogenes (OK-432) that is also a GMP grade agent. Therefore, sufficient amounts of FCS-independent fusions were successfully generated with decreased potential hazards of FCS. The FCS-independent fusions expressed tumor-associated antigens, HLA-DR, costimulatory molecules, IL-12, and IL-10. Stimulation of T cells with fusions from healthy donors resulted in proliferation of T cells with high expression levels of perforin/granzyme B and IFN-gamma and efficient induction of antigen-specific cytotoxic T lymphocytes (CTLs). Selection and expansion of T-cell clones were confirmed by TCR Vbeta analysis. However, fusions from patients with metastatic pancreatic cancer induced increased expression levels of TGF-beta1 in CD4+ CD25high T cells and low levels of CTLs with decreased IFN-gamma production.

Regulation of tumor immunity by tumor/dendritic cell fusions.

The goal of cancer vaccines is to induce antitumor immunity that ultimately will reduce tumor burden in tumor environment. Several strategies involving dendritic cells- (DCs)- based vaccine incorporating different tumor-associated antigens to induce antitumor immune responses against tumors have been tested in clinical trials worldwide. Although DCs-based vaccine such as fusions of whole tumor cells and DCs has been proven to be clinically safe and is efficient to enhance antitumor immune responses for inducing effective immune response and for breaking T-cell tolerance to tumor-associated antigens (TAAs), only a limited success has occurred in clinical trials. This paper reviews tumor immune escape and current strategies employed in the field of tumor/DC fusions vaccine aimed at enhancing activation of TAAs-specific cytotoxic T cells in tumor microenvironment.

In vitro generation of cytotoxic and regulatory T cells by fusions of human dendritic cells and hepatocellular carcinoma cells.

BACKGROUND: Human hepatocellular carcinoma (HCC) cells express WT1 and/or carcinoembryonic antigen (CEA) as potential targets for the induction of antitumor immunity. In this study, generation of cytotoxic T lymphocytes (CTL) and regulatory T cells (Treg) by fusions of dendritic cells (DCs) and HCC cells was examined. METHODS: HCC cells were fused to DCs either from healthy donors or the HCC patient and investigated whether supernatants derived from the HCC cell culture (HCCsp) influenced on the function of DCs/HCC fusion cells (FCs) and generation of CTL and Treg. RESULTS: FCs coexpressed the HCC cells-derived WT1 and CEA antigens and DCs-derived MHC class II and costimulatory molecules. In addition, FCs were effective in activating CD4+ and CD8+ T cells able to produce IFN-gamma and inducing cytolysis of autologous tumor or semiallogeneic targets by a MHC class I-restricted mechanism. However, HCCsp induced functional impairment of DCs as demonstrated by the down-regulation of MHC class I and II, CD80, CD86, and CD83 molecules. Moreover, the HCCsp-exposed DCs failed to undergo full maturation upon stimulation with the Toll-like receptor 4 agonist penicillin-inactivated Streptococcus pyogenes. Interestingly, fusions of immature DCs generated in the presence of HCCsp and allogeneic HCC cells promoted the generation of CD4+ CD25high Foxp3+ Treg and inhibited CTL induction in the presence of HCCsp. Importantly, up-regulation of MHC class II, CD80, and CD83 on DCs was observed in the patient with advanced HCC after vaccination with autologous FCs. In addition, the FCs induced WT1- and CEA-specific CTL that were able to produce high levels of IFN-gamma. CONCLUSION: The current study is one of the first demonstrating the induction of antigen-specific CTL and the generation of Treg by fusions of DCs and HCC cells. The local tumor-related factors may favor the generation of Treg through the inhibition of DCs maturation; however, fusion cell vaccination results in recovery of the DCs function and induction of antigen-specific CTL responses in vitro. The present study may shed new light about the mechanisms responsible for the generation of CTL and Treg by FCs.

[Diagnosis of Helicobacter pylori infection: comparison with gold standard].

Marshall and Warren were the first to succeed in culturing Helicobacter pylori (H. pylori) from the gastric mucosa of patients with gastritis in 1983. H. pylori is a spiral-shaped bacterium that resides in the gastric mucosa and is one of the most common infections worldwide. H. pylori infection causes gastritis and peptic ulcers and is associated with the development of gastric cancer and MALT lymphoma. Now, a variety of accurate diagnostic tests are widely available. Both invasive tests (bacterial culture, histopathology, and RUT) and non-invasive tests (UBT and serological test) are conducted for the diagnosis of H. pylori infection. This review provides a general overview of the diagnostic methods and tests the characteristics (sensitivity and specificity) for H. pylori infection.

Expression of immune checkpoint molecules of T cell immunoglobulin and mucin protein 3/galectin-9 for NK cell suppression in human gastrointestinal stromal tumors.

Monoclonal antibody therapy for immune checkpoint blockade has achieved promising results for several types of malignant tumors. For the future treatment of gastrointestinal stromal tumors (GISTs) by immune checkpoint blockade, expression of immune checkpoint-related molecules that suppress antitumor immunity in GISTs was examined. Infiltration of immune cell types into 19 GIST tissues was analyzed by immunohistochemistry, and expression of T cell immunoglobulin and mucin protein 3 (Tim-3) and programmed cell death-1 (PD-1) in the infiltrated immune cells was examined by immunofluorescence microscopy. The expression status of galectin-9 in the GIST tumor cells was also determined by immunohistochemistry. All the GIST tissues showed CD8+ T cell infiltration and 8 showed CD56+ natural killer (NK) cell infiltration, and the numbers of infiltrated CD8+ T and NK cells were strongly correlated. However, these CD8+ T and NK cells were CD69-negative inactivated cells. Tim-3 was expressed in the infiltrated NK cells in 6/8 (75%) of the GIST tissues. Expression of galectin-9, a ligand of Tim-3, was observed in 13/19 (68.4%) GIST tissues and all of the GIST tissues with Tim-3+ NK cell infiltration showed positive galectin-9 expression. No PD-1 expression in the infiltrated NK cells and neither Tim-3 nor PD-1 expression was observed in the infiltrated CD8+ T cells. Interaction between Tim-3 in infiltrated NK cells and galectin-9 in tumor cells may be involved in an immune checkpoint mechanism for suppression of antitumor immunity in GISTs. Blockade of the Tim-3/galectin-9 pathway may become a new strategy for GIST treatment.

Gemcitabine treatment enhances HER2 expression in low HER2-expressing breast cancer cells and enhances the antitumor effects of trastuzumab emtansine.

Trastuzumab emtansine (T-DM1), trastuzumab-conjugated with a cytotoxic agent, has shown promising antitumor effects in breast cancer. Since a good therapeutic response using T-DM1 treatment requires high human epidermal growth factor receptor 2 (HER2) expression, breast cancers with low or no HER2 expression have not been used for T-DM1 treatment. The aim of the present study was to show that treatment of low HER2-expressing breast cancer cells with gemcitabine (GEM) enhanced HER2 expression using RT-qPCR, immunoblot and flow cytometric analysis. The results showed that GEM treatment significantly enhanced HER2 expression in MDA-MB-231, MCF7 and BT-20 breast cancer cells, while paclitaxel (PTX) treatment induced lower or no enhancement in HER2 expression. The expression of HER2 mRNA was also enhanced in GEM-treated MCF7 cells. Treatment with an inhibitor for nuclear factor-(NF)-κB suppressed GEM-induced HER2 upregulation, indicating that NF-κB activation by GEM may be associated with HER2 upregulation. T-DM1 binding to HER2 on MCF-7 cells was enhanced by GEM pretreatment and the combined treatment of GEM and T-DM1 synergistically inhibited the proliferation of MCF7 cells. Thus, the combined treatment with GEM and T-DM1 may be a promising therapeutic modality for low HER2-expressing breast cancers, which was facilitated by the unique HER2-upregulating effect of GEM.

Production of corticotropin-releasing factor and urocortin from human monocyte-derived dendritic cells is stimulated by commensal bacteria in intestine.

AIM: To examine whether commensal bacteria are a contributing cause of stress-related mucosal inflammation. METHODS: Human peripheral blood monocyte-derived dendritic cells (MoDCs) were stimulated by commensal bacterial strains, including Escherichia coli, Clostridium clostridioforme, Bacteroides vulgatus (B. vulgatus), Fusobacterium varium (F. varium), and Lactobacillus delbrueckii subsp. bulgaricus. After incubation, corticotropin-releasing factor (CRF) and urocortin 1 (UCN1) mRNA in the cells was examined by real-time reverse transcription polymerase chain reaction. Supernatants from the cells were tested for CRF and UCN1 using an enzyme-linked immunosorbent assay. RESULTS: Both CRF and UCN1 were significantly augmented by B. vulgatus and F. varium at both the mRNA and protein levels. In particular, B. vulgatus stimulated human MoDCs, resulting in extremely high levels of CRF and UCN1. CONCLUSION: Stimulation of MoDCs by B. vulgatus and F. varium may be associated with CRF/UCN1-related intestinal disorders, such as irritable bowel syndrome and inflammatory bowel disease.

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.

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/m) 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.

Strategies to improve the immunogenicity of anticancer vaccines based on dendritic cell/malignant cell fusions.

The rationale for fusing dendritic cells (DCs) with whole tumor cells to generate anticancer vaccines resides in the fact that the former operate as potent antigen-presenting cells, whereas the latter express a constellation of tumor-associated antigens (TAAs). Although the administration of DC/malignant cell fusions to cancer patients is safe and this immunotherapeutic intervention triggers efficient tumor-specific T-cell responses in vitro, a limited number of objective clinical responses to DC/cancer cell fusions has been reported thus far. This review discusses novel approaches to improve the immunogenicity of DC/malignant cell fusions as anticancer vaccines.

Fusions between dendritic cells and whole tumor cells as anticancer vaccines.

Various strategies have been developed to deliver tumor-associated antigens (TAAs) to dendritic cells (DCs). Among these, the fusion of DCs and whole cancer cells can process a broad array of TAAs, including hitherto unidentified molecules, and present them in complex with MHC Class I and II molecules and in the context of co-stimulatory signals. DC-cancer cell fusions have been shown to stimulate potent antitumor immune responses in animal models. In early clinical trials, however, the antitumor effects of DC-cancer cell fusions are not as vigorous as in preclinical settings. This mini-review summarizes recent advances in anticancer vaccines based on DC-cancer cell fusions.

Combined TLR2/4-activated dendritic/tumor cell fusions induce augmented cytotoxic T lymphocytes.

Induction of antitumor immunity by dendritic cell (DC)-tumor fusion cells (DC/tumor) can be modulated by their activation status. In this study, to address optimal status of DC/tumor to induce efficient antigen-specific cytotoxic T lymphocytes (CTLs), we have created various types of DC/tumor: 1) un-activated DC/tumor; 2) penicillin-killed Streptococcus pyogenes (OK-432; TLR4 agonist)-activated DC/tumor; 3) protein-bound polysaccharides isolated from Coriolus versicolor (PSK; TLR2 agonist)-activated DC/tumor; and 4) Combined OK-432- and PSK-activated DC/tumor. Moreover, we assessed the effects of TGF-ß1 derived from DC/tumor on the induction of MUC1-specific CTLs. Combined TLR2- and TLR4-activated DC/tumor overcame immune-suppressive effect of TGF-ß1 in comparison to those single activated or un-activated DC/tumor as demonstrated by: 1) up-regulation of MHC class II and CD86 expression on DC/tumor; 2) increased fusion efficiency; 3) increased production of fusions derived IL-12p70; 4) activation of CD4(+) and CD8(+) T cells that produce high levels of IFN-γ; 5) augmented induction of CTL activity specific for MUC1; and 6) superior efficacy in inhibiting CD4(+)CD25(+)Foxp3(+) T cell generation. However, DC/tumor-derived TGF-ß1 reduced the efficacy of DC/tumor vaccine in vitro. Incorporating combined TLRs-activation and TGF-ß1-blockade of DC/tumor may enhance the effectiveness of DC/tumor-based cancer vaccines and have the potential applicability to the field of adoptive immunotherapy.