Dendritic cell-based vaccine for pancreatic cancer in Japan

“Vaccell” is a dendritic cell (DC)-based cancer vaccine which has been established in Japan. The DCs play central roles in deciding the direction of host immune reactions as well as antigen presentation. We have demonstrated that DCs treated with a streptococcal immune adjuvant OK-432, produce interleukin-12, induce Th1-dominant state, and elicit anti-tumor effects, more powerful than those treated with the known DC-maturating factors. We therefore decided to mature DCs by the OK-432 for making an effective DC vaccine, Vaccell. The 255 patients with inoperable pancreatic cancer who received standard chemotherapy combined with DC vaccines, were analyzed retrospectively. Survival time of the patients with positive delayed type hypersensitivity (DTH) skin reaction was significantly prolonged as compared with that of the patients with negative DTH. The findings strongly suggest that there may be “Responders” for the DC vaccine in advanced pancreatic cancer patients. We next conducted a small-scale prospective clinical study. In this trial, we pulsed HLA class II-restricted WT1 peptide (WT1-II) in addition to HLA class?I-restricted peptide (WT1-I) into the DCs. Survival of the patients received WT1-I?and -II pulsed DC vaccine was significantly extended as compared to that of the patients received DCs pulsed with WT1-I?or WT1-II alone. Furthermore, WT1-specific DTH positive patients showed significantly improved the overall survival as well as progression-free survival as compared to the DTH negative patients. The activation of antigen-specific immune responses by DC vaccine in combination with standard chemotherapy may be associated with a good clinical outcome in advanced pancreatic cancer. We are now planning a pivotal study of the Vaccell in appropriate protocols in Japan.

Effect of a cancer vaccine prepared by fusions of hepatocarcinoma cells with dendritic cells

AIM: To prepare a cancer vaccine (H(22)-DC) expressing high levels of costimulatory molecules based on fusions of hepatocarcinoma cells (H(22)) with dendritic cells (DC) of mice and to analyze the biological characteristics and induction of specific CTL activity of H(22)-DC. METHODS: DCs were isolated from murine spleen by metrizamide density gradient centrifugation, purified based on its characteristics of semi-adhesion to culture plates and FcR-,and were cultured in the medium containing GM-CSF and IL-4. A large number of DC were harvested. DCs were then fused with H(22) cells by PEG and the fusion cells were marked with CD11c MicroBeads. The H(22)-DC was sorted with Mimi MACS sorter. The techniques of cell culture, immunocytochemistry and light microscopy were also used to test the characteristics of growth and morphology of H(22)-DC in vitro. As the immunogen, H(22)-DC was inoculated subcutaneously into the right armpit of BALB/C mice, and their tumorigenicity in vivo was observed. MTT was used to test the CTL activity of murine spleen in vivo. RESULTS: DC cells isolated and generated were CD11c+ cells with irregular shape, and highly expressed CD80, CD86 and CD54 molecules. H22 cells were CD11c- cells with spherical shape and bigger volume, and did not express CD80, CD86 and CD54 molecules.H(22)-DC was CD11c+ cells with bigger volume, being spherical, flat or irregular in shape, and highly expressed CD80, CD86 and CD54 molecules, too. H(22)-DC was able to divide and proliferate in vitro, but its activity of proliferation was significantly decreased as compared with H(22) cells and its growth curve was flatter than H(22) cells. After subcutaneous inoculation over 60 days, H(22)-DC showed no tumorigenecity in mice, which was significantly different from control groups (P【0.01). The spleen CTL activity against H(22) cells in mice implanted with fresh H(22)-DC was significantly higher than control groups (P 【 0.01). CONCLUSION: H(22)-DC could significantly stimulate the specific CTL activity of murine spleen, which suggests that the fusion cells have already obtained the function of antigen presenting of parental DC and could present H(22)specific antigen which has not been identified yet, and H(22)-DC could induce antitumor immune response; although simply mixed H(22) cells with DC could stimulate the specific CTL activity which could inhibit the growth of tumor in some degree, it could not prevent the generation of tumor. It shows that the DC vaccine is likely to become a helpful approach in immunotherapy of hepatocarcinoma.

An effective vaccine against colon cancer in mice:Use of recombinant adenovirus interleukin-12 transduced dendritic cells

AIM： To investigate the effect of a vaccine with recombinant adenovirus interleukin-12 （AdVIL-12） transduced dendritic cells （DCs） against colon cancer in mice. METHODS： DCs and AdVIL-12 were incubated together at different time intervals and at different doses. Supernatant was collected and tested for IL-12 by enzyme-linked immunosorbent assay （ELISA）. In order to determine whether tumor cell lysate-pulsed （TP） AdVIL-12/DCs enhance therapeutic potential in the established tumor model, CT26 colon tumor cells were implanted subcutaneously （s.c.） in the midflank of naive BALB/c mice. Tumor-bearing mice were injected with a vaccination of CT26 TP AdVIL-12/DCs on d 3 and 10. As a protective colon tumor model, naive BALB/c mice were immunized s.c. in their abdomens with CT26 TP AdVIL-12/DCs twice at seven day intervals. After the immunization on d 7, the mice were challenged with a lethal dose of CT26 tumor cells and survival times were evaluated. Subsequently, cytotoxic T lymphocyte （CTL） activity and interferon gamma （IFNy） secretion was evaluated in the immunized mice, and assayed CTL ex vivo. RESULTS： Murine DCs were retrovirally transduced with AdVIL-12 efficiency, and the AdVIL-12 transduced DCs secreted a high level of IL-12 （AdVIL-12/DCs, 615.27 ± 42.3 pg/mL vs DCs, 46.32 ± 7.29 pg/mL, P 〈 0.05）. Vaccination with CT26 TP AdVIL-12/DCs could enhance anti-tumor immunity against CT26 colon tumor in murine therapeutic models （tumor volume on d 19：CT26 TP AdVIL-12/DCs 107 ± 42 mm^3 vs CT26 TP DCs 383± 65 mm^3, P 〈 0.05） and protective models. Moreover, the CT26 TP AdVIL-12/DC vaccination enhances tumor-specific CTL activity, producing high levels of IFN7 in immunized mice. Ex vivo primed T cells with AdVIL-12/DCs were able to induce more effective CTL activity than in primed T cells with CT26 TP/DCs （E：T = 100：1, 69.49% ± 6.11% specific lysis vs 37.44% ＋ 4.32% specific lysis, P 〈 0.05）.CONCLUSION： Vaccination with recombinant AdVIL-12 transduced DC pulsed tumor cell lysate enhance antitumor immunity specific to colon cancer in mice.

Advances in inducing adaptive immunity using cell-based cancer vaccines: clinical applications in pancreatic cancer

The incidence of pancreatic ductal adenocarcinoma(PDA) is on the rise, and the prognosis is extremely poor because PDA is highly aggressive and notoriously difficult to treat. Although gemcitabine- or 5-fluorouracil-based chemotherapy is typically offered as a standard of care, most patients do not survive longer than 1 year. Therefore, the development of alternative therapeutic approaches for patients with PDA is imperative. As PDA cells express numerous tumor-associated antigens that are suitable vaccine targets, one promising treatment approach is cancer vaccines. During the last few decades, cell-based cancer vaccines have offered encouraging results in preclinical studies. Cell-based cancer vaccines are mainly generated by presenting whole tumor cells or dendritic cells to cells of the immune system. In particular, several clinical trials have explored cell-based cancer vaccines as a promising therapeutic approach for patients with PDA. Moreover, chemotherapy and cancer vaccines can synergize to result in increased efficacies in patients with PDA. In this review, we will discuss both the effect of cell-based cancer vaccines and advances in terms of future strategies of cancer vaccines for the treatment of PDA patients.

Impact of 5-Fu/oxaliplatin on mouse dendritic cells and synergetic effect with a colon cancer vaccine

Objective： The aim of the present study was to investigate the effects of 5-fluorouracil（5-Fu） and oxaliplatin on the function and activation pathways of mouse dendritic cells（DCs）, and to clarify whether 5-Fu/oxaliplatin combined with the CD1d-MC38/α-galactosylceramide（α-GC） tumor vaccine exhibits synergistic effects on the treatment of colon cancer in mice.Methods： The combination of the Toll like receptor（TLR） ligands and/or 5-Fu/oxaliplatin was added into myeloid-derived DCs in vitro culture. DC phenotypic changes were detected by flow cytometry, and the secretion of DC cytokines was detected by cytometric bead array（CBA）. A MC38 mouse colon cancer model was constructed and the DCs were isolated from the spleen, tumor tissue and lymph nodes following intraperitoneal injection of 5-Fu/oxaliplatin. The cell phenotypes were detected by flow cytometry. The tumor infiltrating leukocytes,splenocytes and lymph node cells were co-cultured with the dead MC38 tumor cells, and the secretion levels of interferon-γ（IFN-γ） were detected. 5-Fu/oxaliplatin combined with our previously developed CD1d-MC38/α-GC tumor vaccine was used to inhibit the growth of MC38 colon cancer in mice, and the tumor growth rate and survival time were recorded.Results： 5-Fu/oxaliplatin exerted no significant effect on the expression of the stimulating phenotypes of DCs in vitro, while it could reduce the expression of programmed death ligand 1/2（PD-L1/L2） and promote interleukin-12（IL-12） secretion by DCs. Furthermore 5-Fu/oxaliplatin was beneficial to the differentiation of T-helper 1（Th1） cells. 5-Fu/oxaliplatin further enhanced the stimulating phenotypic expression of DCs in tumor bearing mice, decreased PD-L1/L2 expression, and specifically activated the lymphocytes. The CD1d-MC38/α-GC tumor vaccine combined with 5-Fu/oxaliplatin could exert a synergistic role that resulted in a significant delay of the tumor growth rate, and an increase in the survival time of tumor bearing mice.Conclusions： 5-Fu/oxaliplatin decreased the expression of the DC inhibitory phenotypes PD-L1/L2, promoted DC phenotypic maturation in tumor bearing mice, activated the lymphocytes of tumor bearing mice, and exerted synergistic effects with the CD1d-MC38/α-GC colon cancer tumor vaccine.

Combinational adenovirus-mediated gene therapy and dendritic cell vaccine in combating well-established tumors

Recent developments in tumor immunology and biotechnology have made cancer gene therapy and immunotherapy feasible. The current efforts for cancer gene therapy mainly focus on using immunogenes, chemogenes and tumor suppressor genes. Central to all these therapies is the development of efficient vectors for gene therapy. By far, adenovirus （AdV）-mediated gene therapy is one of the most promising approaches, as has confirmed by studies relating to animal tumor models and clinical trials. Dendritic cells （DCs） are highly efficient, specialized antigen-presenting cells, and DC- based tumor vaccines are regarded as having much potential in cancer immunotherapy. Vaccination with DCs pulsed with tumor peptides, lysates, or RNA, or loaded with apoptotic/necrotic tumor cells, or engineered to express certain cytokines or chemokines could induce significant antitumor cytotoxic T lymphocyte （CTL） responses and antitumor immunity. Although both AdV-mediated gene therapy and DC vaccine can both stimulate antitumor immune responses, their therapeutic efficiency has been limited to generation of prophylactic antitumor immunity against re-challenge with the parental tumor cells or to growth inhibition of small tumors. However, this approach has been unsuccessful in combating well-established tumors in animal models. Therefore, a major strategic goal of current cancer immunotherapy has become the development of novel therapeutic strategies that can combat well-established tumors, thus resembling real clinical practice since a good proportion of cancer patients generally present with significant disease. In this paper, we review the recent progress in AdV-mediated cancer gene therapy and DC-based cancer vaccines, and discuss combined immunotherapy including gene therapy and DC vaccines. We underscore the fact that combined therapy may have some advantages in combating well-established tumors vis-a-vis either modality administered as a monotherapy.

Targeting hepatitis B virus antigens to dendritic cells by heat shock protein to improve DNA vaccine potency

AIM： To investigate a novel DNA vaccination based upon expression of the HBV e antigen fused to a heat shock protein （HSP） as a strategy to enhance DNA vaccine potency.METHODS： A pCMV-HBeAg-HSP DNA vaccine and a control DNA vaccine were generated. Mice were immunized with these different construct. Immune responses were measured 2 wk after a second immunization by a T cell response assay, CTL cytotoxicity assay, and an antibody assay in C57BL/6 and BALB/c mice. CT26-HBeAg tumor cell challenge test in vivo was Performed in BALB/c mice to monitor anti-tumor immune responses.RESULTS： In the mice immunized with pCMV-HBe-HSP DNA, superior CTL activity to target HBV-positive target cells was observed in comparison with mice immunized with pCMV-HBeAg （44% ± 5% vs 30% ± 6% in E： T 〉 50：1, P 〈 0,05）, ELISPOT assays showed a stronger T-cell response from mice immunized with pCMV-HBe- HSP than that from pCMV-HBeAg immunized animals when stimulated either with MHC class I or class Ⅱ epitopes derived from HBeAg （74% ± 9% vs 31% ± 6%, P 〈 0.01）. ELISA assays revealed an enhanced HBeAg antibody response from mice immunized with pCMV- HBe-HSP than from those immunized with pCMV-HBeAg. The lowest tumor incidence and the slowest tumor growth were observed in mice immunized with pCMV- HBe-HSP when challenged with CT26-HBeAg.CONCLUSION： The results of this study demonstrate a broad enhancement of antigen-specific CD4^＋ helper,CD8^＋ cytotoxic T-cell, and B-cell responses by a novel DNA vaccination strategy. They also proved a stronger antigen-specific immune memory, which may be superior to currently described HBV DNA vaccination strategies for the treatment of chronic HBV infection.

Study on biological characters of SGC7901 gastric cancer cell-dendritic cell fusion vaccines

AIM： To detect the biological characters of the SGC7901 gastric cancer cell-dendritic cell fusion vaccines.METHODS： The suspending living SGC7901 gastric cancer cells and dendritic cells were induced to be fusioned by polyethylene glycol. Pure fusion cells were obtained by selective culture with the HAT/HT culture systems. The fusion cells were counted at different time points of culture and their growth curves were drawn to reflect their proliferative activities. The fusion cells were also cultured in culture medium to investigate whether they could grow into cell clones. MTT method was used to test the stimulating abilities of the fusion cells on T lymphocytes＇ proliferations. Moreover, the fusion cells were planted into nude mice to observe whether they could grow into new planted tumors in this kind of immunodeficiency animals.RESULTS： The fusion cells had weaker proliferative activity and clone abilities than their parental cells. When they were cultured, the counts of cells did not increase remarkably, nor could they grow into cell clones in culture medium. The fusion cells could not grow into new planted tumors after planted into nude mice. The stimulating abilities of the fusion cells on T lymphocytes＇ proliferations were remarkably increased than their parental dendritic cells. CONCLUSION： The SGC7901 gastric cancer cell-dendritic cell fusion vaccines have much weaker proliferative abilities than their parental cells, but they keep strong abilities to irritate the T lymphocytes and have no abilities to grow into new planted tumors in immunodeficiency animals. These are the biological basis for their antitumor biotherapies.

Differential centrifugation enhances the anti-tumor immune effect of tumor lysate-pulsed dendritic cell vaccine against glioblastoma

Objective This study aimed to improve the antitumor immunocompetence of a tumor lysate-pulsed dendritic cell(DC)vaccine through differential centrifugation and provide a theoretical basis for its clinical application in glioblastoma.Methods Peripheral blood mononuclear cells were extracted using Ficoll-Paque PLUS and induced into mature DCs in vitro with a cytokine cocktail.The modified tumor lysate was generated by differential centrifugation.The maturity markers of DCs in each group,namely the modified tumor lysate,tumor lysate,and negative and positive control groups,were assessed using flow cytometry.Furthermore,their ability to stimulate lymphocyte proliferation and in vitro antitumor effects were assessed using Cell Trace TM CFSE.IFN-γsecretion levels were measured with ELISA.Intracellular reactive oxygen species were measured using 2’,7’-dichlorofluorescein diacetate(DCFDA)staining.The results were statistically analyzed using an unpaired Student’s t-test and were considered significant at P<0.05.Results Compared with tumor lysate-pulsed DCs,modified tumor lysate-pulsed DCs had a higher expression of maturity markers:CD1a(7.38±0.53%vs.4.47±0.75%)and CD83(19.81±4.09%vs.9.64±1.50%),were better capable of stimulating lymphocyte proliferation[proliferation index(PI):8.54±0.16 vs.7.35±0.05],secreting IFN-γ,and inducing stronger in-vitro cytotoxic T lymphocyte(CTL)cytotoxicity against glioblastoma cells.In addition,we found that the level of ROS in modified tumor lysate-pulsed DCs was lower than that in tumor lysate-pulsed DCs.Conclusion Differential centrifugation of tumor lysates can improve the antitumor immunocompetence of DC vaccines,and reactive oxygen species may be the key to affecting DC function in the whole tumor lysate.

Vaccination with dendritic cells pulsed with hepatitis C pseudo particles induces specific immune responses in mice

AIM: To explore dendritic cells (DCs) multiple functions in immune modulation. METHODS: We used bone-marrow derived dendritic cells from BALB/c mice pulsed with pseudo particles from the hepatitis C virus to vaccinate naive BALB/c mice. Hepatitis C virus (HCV) pseudo particles consist of the genotype 1b derived envelope proteins E1 and E2, covering a non-HCV core structure. Thus, not a single epitope, but the whole "viral surface" induces immunogenicity. For vaccination, mature and activated DC were injected subcutaneously twice. RESULTS: Humoral and cellular immune responses measured by enzyme-linked immunosorbent assay and interferon-gamma enzyme-linked immunosorbent spot test showed antibody production as well as T-cellsdirected against HCV. Furthermore, T-cell responses confi rmed two highly immunogenic regions in E1 and E2 outside the hypervariable region 1. CONCLUSION: Our results indicate dendritic cells as a promising vaccination model for HCV infection that should be evaluated further.

Clinical and Immunological Effects in Patients with Advanced Non-Small Cell Lung-Cancer after Vaccination with Dendritic Cells Exposed to an Allogeneic Tumor Cell Lysate

Background: We evaluated the clinical and immunological effects of dendritic cell (DC) vaccination of patients with NSCLC. Autologous DCs were pulsed with a MAGE containing allogeneic melanoma cell lysate (MelCancerVac?, Dandrit Biotech,Copenhagen,Denmark). Imiquimod cream, proleukin and celecoxib were used as adjuvants to the vaccines. The objective of the study was to evaluate specific T cell response in vitro by IFNg EliSpot. Secondary objectives were overall survival, response and quality of life (QoL). Results: Twenty-two patients initiated the vaccination program consisting of ten vaccinations. Seven patients remained in stable disease (SD) three months after the first vaccination. After ten vaccinations (six months), four patients still showed SD and continued vaccinations on a monthly basis. These four patients received a total of 12, 16, 26 and 35 vaccinations, respectively. Five patients showed an unexpectedly prolonged survival. The treatment was well tolerated and only minor adverse events were reported. Quality of life did not change during the study period. In four of the seven patients with SD, vaccine-specific T cells were detected by IFNγ EliSpot assays, whereas only one patient with progressive disease (PD) showed vaccine-specific responses. Conclusion: This DC-based vaccine trial has indicated a correlation between vaccine-specific immunity and sustained SD. Furthermore, we observed an unexpectedly prolonged survival in some patients, which may indicate delayed effect of DC vaccination after completion of the treatment. A prospective randomized phase-IIb or -III is needed to further evaluate the use of MelCancerVac? vaccine treatment in patients with progressive NSCLC.

Immune therapy including dendritic cell based therapy in chronic hepatitis B virus infection

Hepatitis B virus （HBV） infection is a global public health problem. Of the approximately 2 billion people who have been infected worldwide, more than 400 million are chronic carriers of HBV. Considerable numbers of chronic HBV carriers suffer from progressive liver diseases. In addition, all HBV carriers are permanent source of this virus. There is no curative therapy for chronic HBV carriers. Antiviral drugs are recommended for about 10% patients, however, these drugs are costly, have limited efficacy, and possess considerable side effects. Recent studies have shown that immune responses of the host to the HBV are critically involved at every stage of chronic HBV infection： （1） These influence acquisition of chronic HBV carrier state, （2） They are important in the context of liver damages, （3） Recovery from chronic HBV-related liver diseases is dependent on nature and extent of HBV-specific immune responses. However, induction of adequate levels of HBV-specific immune responses in chronic HBV carriers is difficult. During the last one decade, hepatitis B vaccine has been administered to chronic HBV carriers as a therapeutic approach （vaccine therapy）. The present regimen of vaccine therapy is safe and cheap, but not so effective. A dendritic cell-based therapeutic vaccine has recently been developed for treating chronic HBV infection. In this review, we will discuss about the concept, scientific logics, strategies and techniques of development of HBV- specific immune therapies including vaccine therapy and dendritic cell-based vaccine therapy for treating chronic HBV infection.

Induction of hepatitis C virus-specific cytotoxic T and B cell responses by dendritic cells expressing a modified antigen targeting receptor

AIM: To find a novel antigen (Ag) presentation strategy to improve the immune responses induced by dendritic cell (DC)vaccine expressing hepatitis C virus (HCV) core antigen (pcDNA3HCV C-Fc) in Balb/c mice (H-2d).METHODS: pcDNA3HCV C-Fc plasmid and eukaryotic expression vector pcDNA3 were injected into mice sc. Immune responses to pcDNA3HCV C-Fc were studied. Meanwhile the effect of pcDNA3HCV C-Fc on anti-translated subcutaneous tumor of SP2/0 cells stably expressing HCV C Ag (SP2/0-HCV C-FC) was also studied. Anti-HCV C in serum was detected by enzyme-linked immunoadsordent assay (ELISA) and HCV specific cytotoxic T lymphocyte (CTL) activity was measured by LDH release assay. After 3 wk of DNA immunization,the cells of SP2/0-HCV C-FC were inoculated into mice subcutaneously and tumor growth was measured every 5 d.The survival rate and living time of mice were also calculated.RESULTS: After 4 wk of DC immunization, the A450 nm values of sera in mice immunized with pcDNA3HCV C-Fc-DC and pcDNA3-DC were 0.56±0.17 and 0.12±0.03 respectively. The antibody titres in mice codeliveried with pcDNA3HCV C-Fc with DC were significantly higher than those of mice injected with pcDNA3-DC. The HCV specific CTL activities in mice coinjected with DC and pcDNA3HCV C-Fc or empty expression vectors were(73.2±3.1) % and (24.4±8.8) %, which were significantly higher than those of mice injected with water.The DC vaccine could evidently inhibit tumor growth, prolong the survival time of mice and improve the survival rate of mice and these effects could be improved by HCV C-Fc (pcDNA3HCV C-Fc) gene codelivered.CONCLUSION: DC vaccine has a strong antigenicity in humoral and cellular immunities, which can be promoted by transduced pcDNA3HCV C-Fc expressing HCV C or Fc.Thus, pcDNA3HCV C-Fc-transduced DCs may be a promising candidate for a CTL-based vaccine against HCV.

Effects of cytotoxic T lymphocytes on hepatoma cell line SMMC-7721 induced by different subsets of dendritic cells in vitro

BACKGROUND: Dendritic cells (DCs) loaded with complex antigen are always used to induce cytotoxic T lymphocytes (CTLs) which have a specific anti-tumor activity. However, CTLs can assault autologous cells induced by DCs loaded with autologous antigen. This study aimed to explore how to weaken the autoimmune reaction induced by DC vaccine by combining mature DC (mDC) activating immunity and immature DC (imDC) leading to immune tolerance to make hepatocellular carcinoma (HCC) vaccine in vitro. METHODS: DC progenitors derived from human peripheral blood were assigned to two groups. One was cultured to mDC and pulsed with frozen-thawed antigen (FTA) of human HCC cell line SMMC-7721 cells (mDC group), and the other was cultured to imDC and pulsed with FTA of human liver cell line L-02 cells (imDC group). The morphology of DCs was monitored and cells phenotypes including HLA-DR, CD80, CD1α, CD83 were assayed by flowcytometry (FCM). The concentrations of interleukin-12 (IL-12) in the supernatant were assayed by ELISA. Methyl thiazolyl tetrazolium (MTT) was used to evaluate T cell proliferation induced by mDC and imDC and the killing rate of CTL induced by mDC and imDC respectively/together on SMMC-7721 and L-02 cells. RESULTS: Compared with the imDC group, the mDC group was characterized by the following: increased secretion of IL-12 (P【0.05); higher expression of HLA-DR, CDla, CD80, CD83; and stronger activity in stimulating proliferation of isogenic T cells (P【0.05). CTL induced by the mDC group had a significant killing response to SMMC-7721 as well as a higher killing rate for L-02 (P】0.05). CTL induced by mDC and imDC together had a higher killing response to SMMC-7721, but a lower killing rate for L-02(P【0.01). CONCLUSIONS: CTL induced by mDC and imDC together has a higher antigen-specific killing response in vitro than that induced by mDC alone. This may be of greater clinical value.

Enhancing Antitumor by Immunization with Fusion of Dendritic Cells and Engineered Tumor Cells

A novel approach for a dentritic cells (DCs) based tumor vaccine was developed for the formation of hybrid engineered J558 after fusion with DCs. To make the hybrid tumor vaccine generate more efficient specific CTL cytotoxicity against wild type tumor cells, we genetically engineered tumor cells with mIL 12 gene prior to the cell fusion. mIL 12 was detected at 870±60 pg/(10 5 cells/ml) in the culture supernatants and the fusion ratio was about 30 % by the co focal microscopic analysis. Vaccination of mice with DCs fused with engineered J558 induced more efficient tumor specific CTL cytotoxicity against wild type tumor cells in vitro and with efficient antitumor immunity in vivo . These results suggest that this approach of using DCs fused with engineered tumor cells could be applied in clinical settings of DCs based cancer vaccines.

Modulation of the immune response by heterogeneous monocytes and dendritic cells in lung cancer

Different subpopulations of monocytes and dendritic cells(DCs)may have a key impact on the modulation of the immune response in malignancy.In this review,we summarize the monocyte and DCs heterogeneity and their function in the context of modulating the immune response in cancer.Subgroups of monocytes may play opposing roles in cancer,depending on the tumour growth and progression as well as the type of cancer.Monocytes can have pro-tumour and anti-tumour functions and can also differentiate into monocyte-derived DCs(moDCs).MoDCs have a similar antigen presentation ability as classical DCs,including cross-priming,a process by which DCs activate CD8 T-cells by crosspresenting exogenous antigens.DCs play a critical role in generating anti-tumour CD8 T-cell immunity.DCs have plastic characteristics and show distinct phenotypes depending on their mature state and depending on the influence of the tumour microenvironment.MoDCs and other DC subsets have been attracting increased interest owing to their possible beneficial effects in cancer immunotherapy.This review also highlights key strategies deploying specific DC subpopulations in combination with other therapies to enhance the anti-tumour response and summarizes the latest ongoing and completed clinical trials using DCs in lung cancer.

GENETICALLY MODIFIED DENDRITIC CELLS INDUCED SPECIFIC CYTOTOXITY AGAINST HUMAN HCC CELLS IN VITRO

Objective: to transduce the tumor associated antigen gene MAGE-1 and/or IL-12 gene into dendritic cells (DC) and to observe the in vitro cytotoxic effect induced by the genetically modified DC against the human hepatocellular carcinoma (HCC) cell line SMMC7721. Methods: the MAGE-1 gene was inserted into the retrovirus vector LXSN to construct the recombinant retrovirus LMSN. The monocyte-derived DCs were transfected at appropriate differentiation stage by LMSN and/or a recombinant adenovirus AdmiL-12, which containing murine IL-12 gene. The control groups included retrovirus LXSN transfected, adenovirus AdBGFP transfected and non-transfected DCs. The MAGE-1 gene expression was identified by western blot and the mIL-12 p70 secretion was detected by ELISA assay. The in vitro cytotoxicities against SMMC7721 induced by genetically modified and control groups of DC were tested by MTT assay. Results: The MAGE-1 expression was detected by a monoclonal antibody in DCs tranfected with LMSN but not in control groups. At 16 h, 24 h and 48 h after transfection with AdmIL-12, the concentration of the mIL-12 p70 in the culture medium was 580pg/106 cells, 960pg/106 cells and 1100pg/106 cells respectively. The mIL-12 p70 secretions were not detected in other groups. The lytic activity (as judged by % lysis) induced by each groups of DC was 94.2±5.2% (LMSN and AdmIL-12 cotransfected group), 78.9±3.6% (LMSN transfected groups), 52.6±9.7% (AdmIL-12 transfected group), 34.7±4.3% (LXSN transfected group), 36.3±3.8% (AdBGFP transfected group) and 3.9±2.0% (non-transfected group) respectively. Except for LXSN transfected and AdBGFP transfected group, the difference of the lytic activities between other groups were statistically significant (P<0.05). Conclusion: The MAGE-1 gene modified DCs can induce relatively specific cytotoxicty against SMMC7721 in vitro and thus suggested that those genetically engineered DCs have the potential to serve as novel vaccine for HCC. Transduction of exogenous IL-12 gene into DCs may further enhance DCs’ activity and the effectiveness of the above DC vaccine.

Construction of HA-1-DC Nucleic-acid Vaccine and Induction of Specific Cytotoxic T Lymphocytes

An HA-1-DC nucleic-acid vaccine was constructed to induce anti-leukemia effect after hematopoietic stem cell transplantation (HSCT). DCs were generated from HSCT donors in vitro, and its immunologic activity was assayed by using flow cytometry and mixed lymphocytes reaction. HA-1 gene was electroporated into the cultured DCs to construct a DC nucleic-acid vaccine. After transfection for 48 h, the expression of HA-1 protein could be detected by using Western blot. The DCs were cultured with syngenic lymphocytes to induce specific cytotoxic T lymphocytes (CTLs). The cytoxicity of the CTLs was detected by LDH assay. The results showed that The DCs derived from peripheral blood monocytes (PBMCs) expressed the phenotype of DCs, and were effective in stimulating proliferation of the allogenic lymphocytes. After electroporating for 48-h, HA-1 protein was detected by using Western blot. The cytotoxity of inducing CTLs was higher than the control group. It was suggested that minor histocompatibility antigen HA-1 could be considered as a target of immunotherapy against leukemia after HSCT.

HER2-Specific Vaccines for HER2-Positive Breast Cancer Immunotherapy

Anti-human epidermal growth factor receptor-2 (HER2) immunization can be elicited by vaccination with DNA encoding the extra- or intra-cellular domain (ECD or ICD) of HER2, naked or encap-sulated in viral vectors. HER2-peptides derived from ECD or ICD of HER2, and HER2-pulsed dendritic cells (DCs) or engineered DCs expressing HER2, respectively. We performed a computer- based literature search which includes but is not limited to the following keywords: breast cancer, immunotherapy, HER2-peptide vaccine, HER2-DNA vaccine, HER-DC vaccine, HER2 vaccine, and HER2/neu, in PubMed, Medline, EMBO and Google Scholar;data from recently reported clinical trials were also included. Drawing upon this synthesis of literature, this work summarizes the de-velopment and current trend in experimental and clinical investigations in HER2-positive breast cancer using HER2-specific vaccine and immunotherapy, focusing especially on: (i) DNA-;(ii) peptide-;and (iii) DC-based vaccines. It addresses interventions that have been applied to overcome immunotolerance thereby to improve treatment outcomes. These include blocking the inhibitory cytotoxic T lymphocyte-associated protein-4 (CTLA-4), which is expressed at high levels by regulatory T (Treg) cells, or complete Treg depletion to improve T-cell activation. Moreover, modulatory interventions can provide further improvement in the efficacy of HER2-specific vaccine. The interventions include the use of immunogenic adjuvants such as cytokines interleukin-12 (IL-12), tumor necrosis factor (TNF)-α and granulocyte-macrophage colony-stimulating factor (GM-CSF), the use of Toll-like receptor (TLR) ligands and tetanus toxin’s universal epitopes such as the CD4+ help T (Th)-epitope P30, and the use of either chimeric or heterogenous xenogeneic HER2. Combining active HER2-vaccination with adoptive trastuzumab antibody immunotherapy is likely to increase the effectiveness of each approach alone. The development of effective HER2-vaccines for breast cancer remains a critical challenge. Though these novel interventions seem promising, further investigation is still needed since the results are preliminary. Furthermore, the review discusses the challenges and future perspectives in HER2-vaccine research and development.

RNA vaccines for anti-tumor therapy

The immune system is able to recognize tumor antigens and this has been the basis for the development of cancer immunotherapies. The immune system can be instructed to recognize and attack tumor cells by means of vaccination strategies. One such strategy involves the delivery of tumor antigen as genetic material. Herewith we describe the use of RNA encoding tumor antigens for vaccination purposes in tumor settings. RNA has features that are interesting for vaccination. Upon transfection, the RNA has no possibility of integration into the genome, and the tumor translated proteins enter the intrinsic antigen processing pathway thus enabling presentation by MHC-I molecules. This can specifically activate cytotoxic CD8 T cells that can attack and kill tumor cells. RNA can be delivered as a naked molecule for vaccination purposes or can be used to transfect dendritic cells. The combination of RNA technology with dendritic cell vaccination provides a powerful tool for cancer immunotherapies.