Antitumor effects of vaccination with dendritic cells transfected with modified receptor for hyaluronan-mediated motility mRNA in a mouse glioma model.

OBJECT: The receptor for hyaluronan-mediated motility (RHAMM) is frequently overexpressed in brain tumors and was recently identified as an immunogenic antigen by using serological screening of cDNA expression libraries. In this study, which was conducted using a mouse glioma model, the authors tested the hypothesis that vaccination with dendritic cells transfected with RHAMM mRNA induces strong immunological antitumor effects. METHODS: The authors constructed a plasmid for transduction of the mRNAs transcribed in vitro into dendritic cells, which were then used to transport the intracellular protein RHAMM efficiently into major histocompatibility complex class II compartments by adding a late endosomal-lysosomal sorting signal to the RHAMM gene. The dendritic cells transfected with this RHAMM mRNA were injected intraperitoneally into the mouse glioma model 3 and 10 days after tumor cell implantation. The antitumor effects of the vaccine were estimated by the survival rate, histological analysis, and immunohistochemical findings for immune cells. Mice in the group treated by vaccination therapy with dendritic cells transfected with RHAMM mRNA survived significantly longer than those in the control groups. Immunohistochemical analysis revealed that greater numbers of T lymphocytes containing T cells activated by CD4+, CD8+, and CD25+ were found in the group vaccinated with dendritic cells transfected with RHAMM mRNA. CONCLUSIONS: These results demonstrate the therapeutic potential of vaccination with dendritic cells transfected with RHAMM mRNA for the treatment of malignant glioma.

Tumor cells loaded with alpha-galactosylceramide induce innate NKT and NK cell-dependent resistance to tumor implantation in mice.

Dendritic cells (DCs) loaded with alpha-galactosylceramide (alpha-GalCer) are known to be active APCs for the stimulation of innate NKT and NK cell responses in vivo. In this study, we evaluated the capacity of non-DCs to present alpha-GalCer in vitro and in vivo, particularly tumor cells loaded with alpha-GalCer (tumor/Gal). Even though the tumor cells lacked expression of CD40, CD80, and CD86 costimulatory molecules, the i.v. injection of tumor/Gal resulted in IFN-gamma secretion by NKT and NK cells. These innate responses to tumor/Gal, including the induction of IL-12p70, were comparable to or better than alpha-GalCer-loaded DCs. B16 melanoma cells that were stably transduced to express higher levels of CD1d showed an increased capacity relative to wild-type B16 cells to present alpha-GalCer in vivo. Three different tumor cell lines, when loaded with alpha-GalCer, failed to establish tumors upon i.v. injection, and the mice survived for at least 6 mo. The resistance against tumor cells was independent of CD4 and CD8 T cells but dependent upon NKT and NK cells. Mice were protected from the development of metastases if the administration of live B16 tumor cells was followed 3 h or 3 days later by the injection of CD1d(high)-alpha-GalCer-loaded B16 tumor cells with or without irradiation. Taken together, these results indicate that tumor/Gal are effective APCs for innate NKT and NK cell responses, and that these innate immune responses are able to resist the establishment of metastases in vivo.

Glycolipid alpha-C-galactosylceramide is a distinct inducer of dendritic cell function during innate and adaptive immune responses of mice.

alpha-Galactosylceramide (alpha-GalCer) is the prototype compound for studying the presentation of glycolipids on CD1d molecules to natural killer T (NKT) lymphocytes. A single i.v. dose of glycolipid triggers a cascade of events involving the production of several cytokines over the course of a day, a short-lived activation of NKT and natural killer (NK) cells, and a more prolonged adaptive T cell immune response if certain antigens are given together with alpha-GalCer. We find that a recently described analogue, alpha-C-galactosylceramide (alpha-C-GalCer), more potently induces these innate and adaptive immune responses in mice. alpha-C-GalCer acts as a more effective trigger for IL-12 and IFN-gamma production, although it minimally elicits IL-4 and TNF-alpha release into the serum. Also, alpha-C-GalCer better mobilizes NKT and natural killer cells to resist B16 melanoma. To help understand these effects, we find that alpha-C-GalCer binds more stably to dendritic cells than alpha-GalCer and that dendritic cells loaded with alpha-C-GalCer induce larger and more long lasting NKT cell responses in vivo. When glycolipid is targeted to dendritic cells in spleen together with antigens in dying cells, such as irradiated tumor cells, alpha-C-GalCer is active as an adjuvant for T cell-mediated immunity at lower doses, just 20 ng per mouse, where it is also able to up-regulate the required CD40L costimulatory molecule on NKT cells. Therefore, alpha-C-GalCer represents a glycolipid that binds more stably to dendritic cells and acts as a more effective link between innate and adaptive immunity in vivo.

Anti-tumor activity of dendritic cells transfected with mRNA for receptor for hyaluronan-mediated motility is mediated by CD4+ T cells.

Receptor for hyaluronan-mediated motility (RHAMM) is overexpressed in various tumors with high frequency, and was recently identified as an immunogenic antigen by serologic screening of cDNA expression libraries. In this study, we explored whether RHAMM is a potential target for dendritic cell (DC) immunotherapy. We constructed a plasmid for transduction of in vitro-transcribed mRNAs into DCs to efficiently transport the intracellular protein RHAMM into MHC class II compartments by adding a late endosomal/lysosomal sorting signal to the RHAMM gene. Immunization of mice with modified RHAMM mRNA-transfected DCs (DC/RHAMM) induced killing activity against RHAMM-positive tumor cells in splenocytes. To examine whether CD4(+) and/or CD8(+) T cells were required for this antitumor immunity, an anti-CD4 or anti-CD8 antibody was administered to mice after immunization with DC/RHAMM. Depletion of CD4(+) T cells significantly diminished the induction of tumor cell-killing activity in splenocytes, whereas CD8(+) T cell depletion had no effect. We then investigated the therapeutic effect of DC/RHAMM in a 3-day tumor model of EL4. DC/RHAMM was administered to mice on days 3, 7 and 10 after EL4 tumor inoculation. The treatment markedly inhibited tumor growth compared to control DCs. Moreover, antibody-mediated depletion of CD4(+) T cells completely abrogated the therapeutic effect of DC/RHAMM, whereas depletion of CD8(+) T cells had no effect. The results of this preclinical study indicate that DCs transfected with a modified RHAMM mRNA targeted to MHC class II compartments can induce CD4(+) T cell-mediated antitumor activity in vivo.

MUC1 peptide vaccination in patients with advanced pancreas or biliary tract cancer.

BACKGROUND: To evaluate the immunogenicity of MUC1 peptide vaccine in advanced pancreatic and bile duct cancers, a phase I clinical trial was conducted. MATERIALS AND METHODS: A 100-mer MUC1 peptide consisting of the extracellular tandem repeat domain and incomplete Freund's adjuvant were subcutaneously administered to 6 pancreatic and 3 bile duct cancer patients at weeks 1, 3 and 5 and doses ranging from 300 to 3000 microg. Circulating intracytoplasmic cytokine-positive CD4+ T cells and anti-MUC1 IgG antibodies were measured before and after vaccination. RESULTS: There were no adverse events, except for mild reddening and swelling at the vaccination site. In 8 patients eligible for clinical evaluation, 7 had progressive disease and 1 stable disease with a tendency for increased circulating anti-MUC1 IgG antibody after vaccination. CONCLUSION: This phase I clinical trial revealed the safety of a vaccine containing 100-mer MUC1 peptides and incomplete Freund's adjuvant.

Therapeutic effect of dendritic cells loaded with a fusion mRNA encoding tyrosinase-related protein 2 and enhanced green fluorescence protein on B16 melanoma.

Dendritic cells (DCs) loaded with messenger RNA (mRNA) have been proposed to be useful for inducing specific cytotoxic T lymphocytes against tumor antigens. It is now also apparent that tumor antigen-specific T cell tolerance limits the efficacy of active immunotherapy. To improve the efficacy of mRNA-loaded DCs, we constructed a fusion mRNA encoding tyrosinase-related protein 2 (TRP2), which has a late endosomal/lysosomal sorting signal and enhanced green fluorescence protein (EGFP), and evaluated its effect in a murine melanoma model. C57BL/6 mice were challenged subcutaneously (s.c.) with 3 x 10(5) B16 tumor cells, and 3 and 10 days later, 3 x 10(5) DCs loaded with mRNA (DC/mRNA) were injected s.c. in the vicinity of the tumor site. Treatment with DC/TRP2 mRNA or DC/TRP2-EGFP mRNA significantly inhibited tumor growth compared to DC/PBS on day 17 after B16 challenge (DC/PBS vs. DC/TRP2 mRNA, p = 0.0411; DC/PBS vs.DC/TRP2-EGFP mRNA, p = 0.0253), whereas no antitumor effect was observed in mice treated with DC/EGFP mRNA or DC/TRP2 peptide. Moreover, the survival rate in mice immunized with DC/TRP2 mRNA or DC/TRP2-EGFP mRNA was significantly improved as compared with that in mice receiving DC/PBS (DC/PBS vs. DC/TRP2 mRNA, p = 0.0228; DC/PBS vs. DC/TRP2-EGFP mRNA, p = 0.0049). Depletion of CD4+ T cells or CD8+ T cells with antibody administration completely abrogated the therapeutic effectiveness of DC/TRP2-EGFP mRNA, suggesting the induction of a T cell immune response against the B16 tumor.

Evaluation of an mRNA lipofection procedure for human dendritic cells and induction of cytotoxic T lymphocytes against enhanced green fluorescence protein.

We utilized an mRNA lipofection procedure in human dendritic cells (DCs) and attempted to induce cytotoxic T lymphocytes (CTLs) against enhanced green fluorescence protein (EGFP). EGFP mRNA was transfected into phytohemagglutinin (PHA)-stimulated lymphocytes or adherent peripheral blood mononuclear cell-derived DCs using a liposomal reagent. Lipofection efficiency was measured by flow cytometry. In PHA-stimulated lymphocytes, increasing concentrations of liposome or mRNA increased EGFP expression levels by up to 64.4%, but caused a decrease in cell viability. A similar trend was also observed in DCs. For 70% DC viability, the concentration of liposomes was 24 microl/ml, and the mRNA concentration was 6 microg/ml. Under these conditions, ELISPOT and (51)Cr release assays were performed on CD8+ T cells stimulated twice with EGFP mRNA-transfected DCs. The number of interferon-gamma-producing cells was increased when the CD8+ T cells were cocultured for 24 h with PHA-stimulated lymphocytes transfected with EGFP mRNA. The level of specific lysis of EGFP mRNA-transfected DCs also increased to approximately 80%, with an effector to target ratio of 40:1. These data suggest that EGFP is immunogenic for human T cells, confirming that our lipofection procedure may be of use for inducing specific CTLs.

Increased expression of MUC1 in advanced pancreatic cancer.

BACKGROUND: MUC1 is associated with tumor invasion and metastasis, and is expressed in pancreatic cancer with a high frequency. This study explored whether MUC1 expression affected the survival of patients with pancreatic cancer. METHODS: Tissue specimens obtained from 70 patients with invasive ductal carcinoma of the pancreas, in pTNM stage III or IV, were immunostained with the anti-MUC1 monoclonal antibody DF3. The results of immunostaining were determined to be positive when more than 50% of the total cancer cells were positively stained. Association of the expression of the DF3 epitope with clinicopathological parameters or patients' survival was statistically evaluated. RESULTS: The incidence of positivity of MUC1 expression was 55.7% (39/70) and this incidence was significantly higher in pTNM stage IV than in stage III (odds ratio [OR], 4.015; 95% confidence interval [CI], 1.459-11.0541; P = 0.0076). As there was a clear difference in overall survival between pTNM stages III and IV ( P = 0.0016), the effect of MUC1 expression on survival was separately evaluated in each stage. It was shown that the expression of MUC1 was associated with unfavorable overall survival in stage IV ( P = 0.0197). CONCLUSIONS: Our data suggest that the expression of MUC1 may be related to the progression of pancreatic cancer.

Circulating anti-MUC1 IgG antibodies as a favorable prognostic factor for pancreatic cancer.

MUC1 is immunogenic in vivo and humoral and cellular immune responses against MUC1 have been detected in cancer patients. Our study explored the association of circulating anti-MUC1 antibodies with clinicopathological parameters or patients' survival of pancreatic cancer. Serum specimens from 36 patients with invasive ductal carcinoma of the pancreas were subjected to enzyme immunoassay for anti-MUC1 IgG or IgM antibodies. Serum levels of anti-MUC1 IgG antibodies were significantly correlated with survival time (p = 0.0004), whereas neither those of anti-MUC1 IgM nor anti-Galalpha(1,3)Gal IgG antibodies, the latter known as natural antibodies cross-reactive with MUC1, showed a given tendency. Some patients' sera with the higher antibody titer showed the reactivity with MUC1-transfectants of cultured pancreatic cancer cells, but not with MUC1-negative parental cells. When the samples were tentatively divided into 2 groups by the serum level of anti-MUC1 IgG antibodies, the survival of patients was significantly longer in the group with optical density >or=0.3 than in that with optical density <0.3 (p = 0.008). Circulating anti-MUC1 IgG antibody levels remained significant (HR, 0.03; 95% CI, 0.003-0.289; p = 0.0024) after multivariate analysis for pTNM stage, patient age and gender. These data suggest that circulating anti-MUC1-IgG antibody levels may be predictive for survival of pancreatic cancer patients.

Expression of CD66a in multiple myeloma.

CD66a is a member of the carcinoembryonic antigen family and has been suggested to function as an intercellular adhesion molecule and cell growth regulator. Expression of CD66a in myeloma cells was examined with mAb TS135 against CD66a transfectants of murine-transformed fibroblasts. The reactivity of mAb TS135 with CD66a, CD66c, and CD66e was revealed. CD66a in myeloma cells was considered to be detectable with this mAb, since CD66c and CD66e are not expressed in them. CD66a was detected in three myeloma cell lines and an IgM-producing B-cell line. In clinical bone marrow specimens, including 18 multiple myeloma, two primary macroglobulinemia, and a case of CLL-like chronic lymphoproliferation with monoclonal IgG production, CD66a and three conventional myeloma cell markers (PCA-1, CD38, and CD56) were examined by indirect immunofluorescence assay. The results showed that 18 out of 21 cases (86%) were CD66a+, and PCA-1 showed the highest correlation with CD66a among conventional markers. Primary macroglobulinemia and chronic lymphoproliferation were also CD66a+. Two-dimensional flow cytometry with mAbs TS135 and CD38 confirmed the reactivity of TS135 with myeloma cells in those bone marrow specimens. The findings suggest that CD66a is expressed in multiple myeloma with high frequency.