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Regulatory T cells (T(reg) cells) suppress autoreactive immune responses and limit the efficacy of tumor vaccines; however, it remains a challenge to selectively eliminate or inhibit T(reg) cells. In this study, the zinc-finger A20, a negative regulator of the Toll-like receptor and tumor necrosis factor receptor signaling pathways, was found to play a crucial part in controlling the maturation, cytokine production and immunostimulatory potency of dendritic cells (DCs). A20-silenced DCs showed spontaneous and enhanced expression of costimulatory molecules and proinflammatory cytokines and had different effects on T cell subsets: they inhibited T(reg) cells and hyperactivated tumor-infiltrating cytotoxic T lymphocytes and T helper cells that produced interleukin-6 and tumor necrosis factor-alpha and were refractory to T(reg) cell-mediated suppression. Hence, this study identifies A20 as an antigen presentation attenuator in control of antitumor immune responses during both the priming and the effector phases and provides a strategy to overcome T(reg) cell-mediated suppression in an antigen-specific manner, reducing the need to directly target T(reg) cells.

Dendritic cells (DCs) play a pivotal role in T cell-mediated immunity and have been shown to induce strong anti-tumor immune responses. As of yet, only a limited number of objective tumor regressions have been observed in clinical studies using a DC vaccine. Suppressor of cytokine signaling-1 (SOCS1) is a key negative regulator of the JAK/STAT signal pathway and plays an essential role in suppressing systemic autoimmunity that is mediated by DCs. The aim of this study was to investigate whether SOCS1-silenced DCs can break the vaccine-induced immune tolerance stimulated by high-dose DC, thereby enhancing anti-tumor activity. In the mouse melanoma model, we found that a 2x106 TRP2-pulsed DC vaccine was able to induce immune tolerance, while a 2x106 SOCS1-silenced DC/TRP2 vaccine prevented immune tolerance. Further experiments revealed that activation-induced T cell death (AICD) through the Fas/Fas-L pathway may play a crucial role in immune tolerance induced by 2x106 TRP2-pulsed DC. SOCS1-silencing in DCs could prevent immune tolerance by inhibiting Fas and Fas-L expression, induced by an increase in IL-12p70 and IL-6 production. In addition, in 2x106 SOCS1-silenced DC/TRP2 immunized mice, higher levels of IL-12p70 and IFN-γ and lower IL-17 production may inhibit tumor angiogenesis and therefore assist in breaking immune tolerance. In conclusion, high-doses of DCs can inhibit the vaccine-induced AICD of T cells and cytokine regulation in tumor angiogenesis. These results indicate that SOCS1-silenced DC vaccines may greatly enhance anti-tumor activity by breaking self-tolerance.

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.

Background & Objective: The abscopal effect on the tumors is a distant antitumor activity induced by local treatments. The study was to observe the induction of abscopal effect by the combination of H101 oncolytic virotherapy with local heating., Methods: Five patients with histologically confirmed, surgically unresectable metastatic malignant tumors (2 nasopharyngeal carcinomas, 1 pulmonary carcinoma, 1 parosteal sarcoma and 1 bladder carcinoma) that had definitely failed to the conventional chemotherapy and radiotherapy or refused these therapies were enrolled in this experimental therapy. All patients were treated with local intra tumor injection of H101 (5x10(11) - 15x10(11) VP) combined with 60-min heating at 42 degrees C., Results: Two patients were cured with complete regressions of both injected and non-injected tumors and have survived for a long period up to date. Three patients responded to the novel therapy variously and eventually died from the disease, who survived 29, 15 and 13 months, respectively., Conclusion: The abscopal antitumor effect could be induced by the combination of H101 local intratumoral injection with heating.

Breast cancer, the most common malignancy in women, has a known association with the steroid hormone estrogen. Estrogen receptor alpha (ERalpha) plays an important role in the clinical care of breast cancer patients, both as a prognostic factor and as a therapeutic target. Here, we show that a small interfering RNA (siRNA) against ERalpha downregulates ERalpha expression in human MCF-7 and Bcap-37 breast cancer cells, causing a significant decrease in breast cancer cell proliferation. Tumor cells lacking ERalpha expression grew at a much slower rate than did control cells in vitro. Moreover, ERalpha knockdown in breast cancer cells resulted in decreased, even completely abrogated tumor growth in BALB/c nude mice, providing direct evidence for an essential role of ERalpha in breast cancer growth. Our results suggest siRNA-mediated gene silencing of ERalpha may impair tumorigenicity, and even suppress the tumor growth.

Dendritic cell (DC)-based tumor vaccines are being extensively tested to treat cancer patients. Although the results of most DC-based clinical trials have been disappointing, recent advances in the basic molecular understanding of positive and negative regulation of antigen presentation and immune responses can form a basis to enhance the efficacy of DC-based vaccines. Here we describe the new understanding of the importance of Toll-like receptor, tumor necrosis factor receptor, and cytokine receptor signaling in activation of innate and adaptive immunity. In particular, we describe the emerging importance of hardwired negative regulators, such as cytokine signaling regulators, as antigen presentation attenuators (APAs), providing a new strategy to break self-tolerance and enhance the potency of tumor vaccines by inhibiting APAs.

Antigen-presenting cells (APCs), such as dendritic cells (DCs), play a critical role in initiating and regulating immune responses. The mechanisms utilized by APCs to sense microbes and initiate immune responses have been well studied, however, little is known about the negative regulation of APCs. Recent studies demonstrate that suppressor of cytokine signaling 1 (SOCS1) functions as an antigen-presentation attenuator by controlling the tolerogenic state of DCs and the magnitude of antigen presentation. Inhibition of SOCS1 significantly enhances the ability of DC-based tumor vaccines to break self-tolerance and to induce effective antitumor immunity. Furthermore, inhibition of this antigen-presentation attenuator drastically boosts memory HIV-specific T- and B-cell responses induced by DC- and DNA-based HIV vaccines. This novel immunization strategy of 'inhibiting antigen-presentation attenuators' would be generally applicable to enhance both therapeutic and prophylactic vaccines against tumors and pathogens.

Gene modification of tumor cells is commonly utilized in various strategies of immunotherapy preventive both as treatment and a means to modify tumor growth. Gene transfer prior to surgery as neoadjuvant therapy has not been studied systematically. We addressed, whether direct intra-tumoral injection of a recombinant adenovirus expressing the immunomodulatory molecule, heat shock protein 72 (ADHSP72), administered prior to surgery could result in sustainable anti-tumor immune responses capable of affecting tumor progression and survival in a number of different murine and rat tumor models. Using intra-dermal murine models of melanoma (B16), colorectal carcinoma (CT26), prostate cancer (TrampC2) and a rat model of glioblastoma (9L), tumors were treated with vehicle or GFP expressing adenovirus (ADGFP) or ADHSP72. Tumors were surgically excised after 72 h. Approximately 25-50% of animals in the ADHSP72 treatment group but not in control groups showed sustained resistance to subsequent tumor challenge. Tumor resistance was associated with development of anti-tumor cellular immune responses. Efficacy of ADHSP72 as neoadjuvant therapy was dependent on the size of the initial tumor with greater likelihood of immune response generation and tumor resistance associated with smaller tumor size at initial treatment. ADHSP72 neoadjuvant therapy resulted in prolonged survival of animals upon re-challenge with autologous tumor cells compared to ADGFP or vehicle control groups. To study the effects on tumor progression of distant metastases, a single tumor focus of animals with multifocal intra-dermal tumors was treated. ADHSP72 diminished progression of the secondary tumor focus and prolonged survival, but only when the secondary tumor focus was <50 mm3 . Our results indicate that gene modification of tumors prior to surgical intervention may be beneficial to prevent recurrence in specific circumstances.

Tumor vaccines represent a promising therapeutic approach, but thus far have achieved only limited success in the clinic. The major challenge is to find a means of overcoming inhibitory immune regulatory mechanisms and eliciting effective T-cell responses to antigens preferentially expressed by tumor cells. Here we show that the stimulatory capacity of dendritic cells (DCs) and the magnitude of adaptive immunity are critically regulated by the suppressor of cytokine signaling (SOCS) 1 in DCs. Silencing SOCS1 in antigen-presenting DCs strongly enhances antigen-specific anti-tumor immunity. Our findings indicate that SOCS1 represents an inhibitory mechanism for qualitatively and quantitatively controlling antigen presentation by DCs and the magnitude of adaptive immunity. This study has implications for understanding the regulation of antigen presentation and for developing more effective tumor vaccines by silencing the critical brake in antigen presentation.

Tumor cells harbor a repertoire of unique, mutated antigens and shared self-antigens but generally are incapable of provoking an effective immune response, likely because of inadequate antigen presentation by professional antigen-presenting cells. Heat shock proteins (HSPs) play important roles in eliciting innate and adaptive immunity by chaperoning peptides for antigen presentation and providing endogenous danger signaling. Although effective in inducing tumor-specific immunity in mice and in some clinical trials, tumor-derived HSPs have many limitations like vaccines, such as the technical difficulty of ex vivo preparation of adequate quantities of HSPs from the resected tumors of individual patients. Here we have developed an in vivo HSP-suicide gene tumor vaccine by generating a recombinant replication-defective adenovirus (Ad-HT) that coexpresses HSP70 and a herpes simplex virus thymidine kinase suicide gene. The combination of HSP70 overexpression in situ and tumor killing by thymidine kinase/ganciclovir treatment, but neither strategy alone, provoked potent systemic antitumor activities after intratumor injection of Ad-HT. Tumor-specific CD4+ and CD8+ T-cell responses were induced by Ad-HT intratumor injection. CD11c+ dendritic cells (DCs) isolated from mice treated with Ad-HT were able to prime tumor-specific CTLs. Collectively, these results indicate that the combination of tumor killing by activation of a suicide gene to release tumor antigens and in situ HSP70 overexpression to enhance DC antigen presentation overcomes host immune tolerance to tumor antigens, leading to the induction of potent antitumor immunity. Our findings may have broad relevance to the use of the in vivo HSP/suicide gene tumor vaccine in therapy for human solid tumors.

Purpose: The carcinoembryonic antigen (CEA) is extensively expressed on the vast majority of colorectal, gastric, and pancreatic carcinomas, and, therefore, is a good target for tumor immunotherapy. CD4+ T-helper (Th) cells play a critical role in initiation, regulation, and maintenance of immune responses. In this study, we sought to identify Th epitopes derived from CEA which can induce CEA-specific Th responses. The combined application with cytotoxic T lymphocyte (CTL) epitopes would be more potent than tumor vaccines that primarily activate CTL alone., Methods: We utilized a combined approach of using a computer-based algorithm analysis TEPITOPE and in vitro biological analysis to identify Th epitopes in CEA., Results: Initial screening of healthy donors showed that all five predicted peptides derived from CEA could induce peptide-specific T-cell proliferation in vitro. We characterized these CEA epitopes by establishing and analyzing peptide-specific T-cell clones. It was shown that CD4+ T-cells specific for the CEA(116 )epitope can recognize and respond to naturally processed CEA protein and CEA(116 )epitope can be promiscuously presented by commonly found major histocompatibility complex (MHC) alleles. Furthermore, it was demonstrated that immunization of human leukocyte antigen (HLA)-DR4 transgenic mice with CEA(116) peptide elicited antigen-specific Th responses which can recognize the antigenic peptides derived from CEA protein and CEA-positive tumors., Conclusion: The MHC class II-restricted epitope CEA(116) could be used in the design of peptide-based tumor vaccine against several common cancers expressing CEA.

Dendritic cells (DCs) are potent antigen-presenting cells (APCs) that play a pivotal role in stimulating antigen-specific T cells in vivo. The cardinal properties of DCs are: the ability to take up, process, and present antigens; (2) the ability to migrate through different tissues into lymphoid organs; and (3) the ability to interact with and stimulate T cells (3). Because of their unique capability of generating primary CD4+ and CD8+ T-cell responses, DCs are of particular interest for immunotherapeutic approaches to infectious disease and cancer. There are many ways to load DCs with antigen and to subsequently induce specific immune responses in vivo and in vitro. One strategy relies on genetically modified peripheral blood mononuclear cell (PBMC)-derived DCs to establish expression of a gene that encodes a specific antigen. In contrast to pulsing DCs with antigenic peptides or proteins, genetic engineering of DCs has the advantages of providing multiple epitopes for major histocompatibility class (MHC) class I-and MHC class II-restricted immune responses as well as a continuous supply of antigen for presentation alone or together with immunomodulatory proteins (cytokines, for example).

Each tumor harbors unique repertoire of mutated antigenic peptides that are immunogenic and potentially can induce tumor-specific immune responses. Because heat shock proteins (HSPs) have the promiscuous ability to chaperone and present a broad repertoire of tumor antigens to antigen presenting cells, HSP tumor vaccine has been tested in clinical trials. However, this vaccine has many limitations, including individual preparation of HSP vaccines from each tumor ex vivo, and quantity of HSPs for therapy strictly limited by the size of the resected tumor mass. Hence, we developed a novel HSP-mediated oncolytic tumor vaccine, referred to as HOT vaccine, by combining the versatile ability of overexpressed HSPs to chaperone antigenic peptides and induce immune responses against a broad array of mutated tumor antigens, with the oncolytic activity of viruses. The results of this study demonstrate that intratumor vaccination with a recombinant oncolytic adenovirus overexpressing the HSP70 protein can eradicate primary tumors, as well as inhibit the growth of established metastatic tumor in mice. Because of its capacity to induce individual tumor-specific immune responses, this HSP-mediated oncolytic tumor vaccine might become a universally applicable, personalized vaccine against any type of solid tumor.

An effective tumor vaccine may require the induction of both CTL and T-helper (Th) cell responses against tumor-associated antigens. Human telomerase reverse transcriptase (hTERT) is highly expressed in >85% of cancer cells and thus is a potential target for tumor vaccines. We therefore sought to identify promiscuous Th epitopes in hTERT, which can be presented by more than one MHC class II allele. Each of 10 peptides derived from hTERT that were predicted to bind to MHC class II molecules was found to be able to induce primary human T-cell responses in vitro. We then established CD4(+) T-cell clones specific for these peptides and found that only hTERT(766) (LTDLQPYMRQFVAHL)-specific CD4(+) Th cells were effective in recognizing naturally processed hTERT antigen. We further found that the naturally processed epitopes hTERT(766) and hTERT(672) (which was identified previously) were promiscuous and capable of inducing CD4(+) T-cell responses in the context of several commonly found HLA-DR alleles, including DR1, DR7, and DR15 for hTERT(672), and DR4, DR11, and DR15 for hTERT(766). We further demonstrated that immunization of humanized HLA-DR4 transgenic mice with hTERT(766) peptide elicited antigen-specific Th responses that can recognize the antigenic peptides derived from hTERT protein and various hTERT-positive tumors, such as breast cancer, melanoma, and leukemia. It was also shown that T-cell precursors specific for the naturally processed epitopes are part of the T-cell repertoires in healthy donors and prostate cancer patients. Thus, these promiscuous, naturally processed Th epitopes in hTERT could be used to develop improved cancer vaccines through the simultaneous stimulation of CTL and Th cells against a broad spectrum of hTERT-positive tumors.

VEGF, an endothelial-specific mitogen, is an important tumor angiogenesis growth factor. The major receptor for VEGF on endothelial cells is KDR. We hypothesized that an intrabody could bind newly synthesized KDR and block receptor transport to the cell surface, thereby inhibiting important VEGF effects. We expressed a single chain antibody (p3S5) to KDR with or without the endoplasmic reticulum (ER) retention signal (KDEL), using either a plasmid (p3S5-HAK) or a tet-off adenoviral system (Ad-HAK). Plasmid-mediated expression of the tethered intrabody significantly reduced KDR expression (from 82.5+/-12.5% to 27.9+/-13.6% of cells; P<0.01) and thymidine incorporation in successfully transfected cells. Ad-HAK infection resulted in intrabody expression in >90% of human umbilical vein endothelial cells (HUVECs), producing marked (80%) apoptosis at 48 h postinfection. The intrabody was essential for these effects, as confirmed by inhibiting its expression with doxycycline or by expressing irrelevant genes (lacZ, GFP). Cell death was dependent on KDR, because Ad-HAK infection of cell lines with minimal or no KDR had little effect on cell viability. Infected HUVECs were unable to form tubes on Engelbreth Holm-Swarm (EHS) tumor gel matrix. These results demonstrate the potential for development of an intrabody-based strategy to block angiogenesis and prevent tumor growth.

An effective tumor vaccine may be required to induce both CTLs and T-helper (Th) responses against tumor-associated antigens. CD4+ Th cells that recognize MHC class II-restricted epitopes play a central role in the initiation and maintenance of antitumor immune responses. Prostate-specific membrane antigen (PSMA) is highly expressed in prostate cancer and thus is a potential target for prostate cancer immunotherapy. In this study, we attempted to identify Th epitopes derived from PSMA for enhancing prostate cancer vaccine by eliciting PSMA-specific Th responses. We first screened a panel of six epitope peptide candidates selected with the TEPITOPE program and found that all six peptides induced peptide-specific T-cell proliferation from one or more donors with estimated T-cell precursor frequencies of 0-4.17 x 10(-6). We then established peptide-specific T-cell clones for five of these six peptides and demonstrated that the T-cell clone specific for the PSMA(459) epitope (NYTLRVDCTPLMYSL) can recognize processed antigens from recombinant PSMA proteins. The PSMA(459) peptide was found to induce CD4+ T-cell responses in healthy individuals and prostate cancer patients with different HLA-DR alleles. To test the potential clinical application, human HLA-DR4 transgenic mice were immunized with PSMA(459) peptide and we found that PSMA(459) peptide immunization activated T cells that specifically responded to antigenic peptides derived from PSMA proteins and PSMA-positive tumor. Thus, the naturally processed Th epitope PSMA(459) could be included in prostate tumor vaccines to enhance PSMA-specific CTL responses.

In this study, a novel approach to antitumor therapy was devised by generating a chimeric tumor-targeted killer protein, referred to as immunocasp-3, that comprises a single-chain anti-erbB2/HER2 antibody with a NH(2)-terminal signal sequence, a Pseudomonas exotoxin A translocation domain, and a constitutively active caspase-3 molecule. In principle, cells transfected with the immunocasp-3 gene would express and secrete the chimeric protein, which then binds to HER2-overexpressing tumor cells. Subsequent cleavage of the constitutively active capase-3 domain from the immunocasp-3 molecule and its release from internalized vesicles would lead to apoptotic tumor cell death. To test this strategy, we transduced human lymphoma Jurkat cells with a chimeric immunocasp-3 gene expression vector and showed that they not only expressed and secreted the fusion protein but also selectively killed tumor cells overexpressing HER2 in vitro. i.v. injection of the transduced Jurkat cells led to tumor regression in a mouse xenograft model because of continuous secretion of immunocasp-3 by the transduced cells. The growth of HER2-positive tumor cells in this model was inhibited by i.m. as well as intratumor injection of immunocasp-3 expression plasmid DNA, indicating that the immunocasp-3 molecules secreted by transfected cells have systematic antitumor activity. We conclude that the immunocasp-3 molecule, combining the properties of a tumor-specific antibody with the proapoptotic activity of a caspase, has potent and selective antitumor activity, either as cell-based therapy or as a DNA vaccine. These findings provide a compelling rationale for therapeutic protocols designed for erbB2/HER2-positive tumors.

CD4+ T cells play critical roles in initiating, regulating, and maintaining antitumor immune responses. One way to improve current tumor vaccines that mainly induce CTLs would be to activate antigen-specific CD4+ T cells that recognize MHC class II restricted tumor associated antigens. Human telomerase reverse transcriptase (hTRT) is preferentially expressed by various tumors and, therefore, could be a universal tumor antigen. In this study, we used a combined approach of using the prediction software TEPITOPE to select class II epitope candidates and in vitro T-cell biological analysis to identify class II-restricted epitope(s) in hTRT. We first identified several HLA-DR7-restricted class-II epitope candidates in hTRT by examining human T-cell responses to synthetic peptides. We then characterized these HLA-DR7-restricted hTRT epitope candidates by establishing and analyzing peptide-specific T-cell clones. It was demonstrated that CD4+ T cells specific for the HLA-DR7-restricted hTRT(672) epitope (RPGLLGASVLGLDDI) can respond to naturally processed hTRT proteins. Furthermore, the hTRT(672)-specific T cells recognized hTRT antigen from various tumors, including prostate cancer, breast cancer, melanoma, and leukemia. Thus, the identification of the naturally processed HLA-DR7-restricted hTRT epitope, together with the previous finding of class I-restricted hTRT epitopes, provide a basis for the combined application of class I- and II-restricted hTRT epitopes to induce potent, long-term CD4+ and CD8+ T-cell responses against a broad spectrum of tumors.

The MYCN oncogene is amplified in approximately 25% of neuroblastoma tumors and is the most significant negative prognostic factor. The direct transcriptional targets of MYCN in MYCN-amplified tumors have not been defined. Microarray analysis of RNA from neuroblastoma primary cell cultures revealed 10-fold higher MCM7 expression in MYCN-amplified versus nonamplified tumors. MCM7 is an essential component of DNA replication licensing factor, a hexameric protein complex that regulates DNA synthesis during the cell cycle, preventing rereplication and ensuring maintenance of DNA euploidy. Additional experiments demonstrated markedly increased expression of MCM7 RNA and protein in MYCN-amplified neuroblastoma tumors and cell lines. Induction of MYCN in conditional cell lines results in increased expression of endogenous MCM7 mRNA and a 3-fold increase in protein levels. In addition, luciferase activity from MCM7 promoter/luciferase gene reporter constructs was significantly increased under MYCN-induced conditions. Specific electrophoretic mobility shifts of MCM7 promoter sequences are detected in extracts of MYCN-amplified cells. These findings demonstrate that in neuroblastoma, the MYCN oncogene directly activates genes required for DNA replication, and this may contribute to neoplastic transformation of these MYCN-amplified tumors.