Induction Of Tumor-specific Immune Responses Research Articles

Induction of tumor-specific immune responses by the TLR9 agonist EnanDIM in murine syngeneic tumor models

Induction of tumor-specific immune responses by the TLR9 agonist EnanDIM in murine syngeneic tumor models

Preclinical evaluation of mRNA trimannosylated lipopolyplexes as therapeutic cancer vaccines targeting dendritic cells

Clinical trials with direct administration of synthetic mRNAs encoding tumor antigens demonstrated safety and induction of tumor-specific immune responses. Their proper delivery to dendritic cells (DCs) requires their protection against RNase degradation and more specificity for dose reduction. Lipid-Polymer-RNA lipopolyplexes (LPR) are attractive mRNA delivery systems and their equipment with mannose containing glycolipid, specific of endocytic receptors present on the membrane of DCs is a valuable strategy. In this present work, we evaluated the capacity of LPR functionalized with a tri-antenna of α-d-mannopyranoside (triMN-LPR) concerning (i) their binding to CD209/DC-SIGN and CD207/Langerin expressing cell lines, human and mouse DCs and other hematopoietic cell populations, (ii) the nature of induced immune response after in vivo immunization and (iii) their therapeutic anti-cancer vaccine efficiency. We demonstrated that triMN-LPR provided high induction of a local inflammatory response two days after intradermal injection to C57BL/6 mice, followed by the recruitment and activation of DCs in the corresponding draining lymph nodes. This was associated with skin production of CCR7 and CXCR4 at vaccination sites driving DC migration. High number of E7-specific T cells was detected after E7-encoded mRNA triMN-LPR vaccination. When evaluated in three therapeutic pre-clinical murine tumor models such as E7-expressing TC1 cells, OVA-expressing EG7 cells and MART-1-expressing B16F0 cells, triMN-LPR carrying mRNA encoding the respective antigens significantly exert curative responses in mice vaccinated seven days after initial tumor inoculation. These results provide evidence that triMN-LPR give rise to an efficient stimulatory immune response allowing for therapeutic anti-cancer vaccination in mice. This mRNA formulation should be considered for anti-cancer vaccination in Humans.

Abstract 4709: Induction of enhanced tumor-specific immunity by Hsp90 targeted photodynamic therapy (Hsp90-PDT) combined with immune checkpoint inhibition

Abstract Background: Immunotherapy has become an emerging anti-cancer therapy, as immune checkpoint blockade with PD-1 or PD-L1 inhibition have been active against multiple cancer types. Nonetheless, there remain many non-responders, suggesting that combinations may improve activity, particularly combinations which can target tumors and have low toxicity. Methods & Results: We have developed a novel Hsp90 targeted photodynamic therapy (Hsp90-PDT), which causes local anti-tumor responses. Hsp90-PDT can be combined with immune checkpoint blockade to generate potent anti-tumor effects. We synthesized well characterized PDT agent, verteporfin (VP) tethered to a HSP90 small molecule inhibitor, to create a Hsp90-PDT agent (Hsp90i-VP). We first compared the uptake of VP and Hsp90i-VP into the breast cancer cell lines (E0771-OVA) in vitro. Hsp90i-VP showed stronger incorporation than VP when the compounds were added 3μM or less in concentration. We next performed in vitro and vivo PDT with Hsp90i-VP and laser exposure (690 nm wavelength). Killing efficiency of VP and Hsp90i-VP was analyzed by MTT assay, which showed increased killing in a dose dependent manner for both photosensitizers (0-30µM) and laser (0-120J/cm2). Next, we treated E0771-OVA tumor-bearing C57BL/6 mice with Hsp90i-VP (25nmol/mouse) administration alone, laser irradiation (240J/cm2) alone, or the combination of Hsp90i-VP and laser. Suppression of the tumor growth was confirmed only in Hsp90i-VP and laser group. In addition, induction of tumor-specific immune response by PDT was confirmed by ELISA, where the elevation of anti-OVA antibody was seen only in PDT group. Furthermore, combination treatment of Hsp90-PDT and anti-PD-L1 mAbs was able to enhance cytotoxic T-cell response and suppress tumor growth significantly even with relatively PDT-resistant cell line. Conclusions: Hsp90-PDT showed a significant ability to induce antigen-specific immune response in both in vitro and in vivo. These results suggest that Hsp90-PDT can play an important role in anti-cancer immune therapy. Our future plan is to investigate the effect of local photodynamic therapy combined with various types of checkpoint inhibitor in breast cancer models. Citation Format: Kensuke Kaneko, Takuya Osada, Timothy A. Haystead, Michael A. Morse, Herbert K. Lyerly. Induction of enhanced tumor-specific immunity by Hsp90 targeted photodynamic therapy (Hsp90-PDT) combined with immune checkpoint inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4709. doi:10.1158/1538-7445.AM2017-4709

A short CD3/CD28 costimulation combined with IL-21 enhance the generation of human memory stem T cells for adoptive immunotherapy.

BackgroundImmunotherapy based on the adoptive transfer of gene modified T cells is an emerging approach for the induction of tumor-specific immune responses. Memory stem T cells, due to their enhanced antitumor and self-renewal capacity, have become potential candidate for adoptive T cell therapy of cancer. Methods to generate memory stem T cells ex vivo rely on CD3/CD28 costimulation and the use of cytokines such as IL-7 and IL-15 during the entire culture period. However, a strong costimulation may induce differentiation of memory stem T cells to effector memory T cells. Here we show that manipulation of the length of the costimulation and addition of IL-21 enhance the ex vivo expansion of memory stem T cells.MethodsPurified naïve T cells from healthy donors were cultured in the presence of anti-CD3/CD28 coated beads, IL-7, IL-15 and/or IL-21 (25 ng/ml). T cells phenotype from the different memory and effector subpopulations were analyzed by multiparametric flow cytometry.ResultsA short anti-CD3/CD28 costimulation of naïve T cells, combined with IL-7 and IL-15 significantly increased the frequencies of CD4+ and CD8+ memory stem T cells ex vivo, compared to a prolonged costimulation (34.6 ± 4.4 % vs 15.6 ± 4.24 % in CD4+; p = 0.008, and 20.5 ± 4.00 % vs 7.7 ± 2.53 % in CD8+; p = 0.02). Moreover, the addition of IL-21 to this condition further enhanced the enrichment and expansion of CD4+ and CD8+ memory stem T cells with an increase in the absolute numbers (0.7 × 106 ± 0.1 vs 0.26 × 106 ± 0.1 cells for CD4+; p = 0.002 and 1.1 × 106 ± 0.1 vs 0.27 × 106 ± 0.1 cells for CD8+; p = 0.0002; short + IL-21 vs long).ConclusionsThese new in vitro conditions increase the frequencies and expansion of memory stem T cells and may have relevant clinical implications for the generation of this memory T cell subset for adoptive cell therapy of patients with cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-016-0973-y) contains supplementary material, which is available to authorized users.

Abstract B057: Optimizing the high throughput in vitro priming of tumor specific T cells

Abstract Peptide-based vaccines are a promising strategy for immunotherapy against cancer. Our group has characterized large numbers of HLA ligands from tumor cells; among them many that might be used for in vivo induction of tumor-specific immune responses. For the development of peptide-based immunotherapies such peptides should be able to induce in vivo tumor-directed immune responses, especially by CD8+ T cells that have a key role in inducing death of tumor cells. The aim of our work is to establish a fast and efficient workflow for immunogenicity testing of tumor-extracted HLA ligands. T cells usually depend on professional antigen presenting cells such as autologous dendritic cells (DCs) for specific induction and expansion. Since the generation of dendritic cells is expensive and time consuming with varying quality and amount of differentiated DCs, we established an artificial system to replace cell-based in vitro priming of T cells, streptavidin-coated artificial antigen presenting cells loaded with defined amounts of recombinant HLA molecules and costimulatory antibodies such as anti-CD28. Using the standard protocol, CD8+ T cells are stimulated three times with artificial antigen presenting cells in weekly intervals in the presence of IL-2 and IL-12. Peptide-specific CD8+ T cells are identified by tetramer staining. Our group started to vary different parameters of this protocol to increase the frequency and number of antigen specific T cells. Alternate costimulatory antibodies and cytokines proved successful to reach that goal. Employing artificial APCs for in vitro priming of tumor-reactive T cells will foster immunogenicity analysis of novel peptides for immunotherapy. Citation Format: Lea Prokop, Stefanie Souczek, Janet Peper, Hans-Georg Rammensee, Stefan Stevanović. Optimizing the high throughput in vitro priming of tumor specific T cells. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B057.

Adaptive Resistance to Immunotherapy Directed Against p53 Can be Overcome by Global Expression of Tumor-Antigens in Dendritic Cells.

Immunotherapy of cancer utilizes dendritic cells (DCs) for antigen presentation and the induction of tumor-specific immune responses. However, the therapeutic induction of anti-tumor immunity is limited by tumor escape mechanisms. In this study, immortalized dendritic D2SC/1 cells were transduced with a mutated version of the p53 tumor suppressor gene, p53M234I, or p53C132F/E168G, which are overexpressed in MethA fibrosarcoma tumor cells. In addition, D2SC/1 cells were fused with MethA tumor cells to generate a vaccine that potentially expresses a large repertoire of tumor-antigens. Cellular vaccines were transplanted onto Balb/c mice and MethA tumor growth and anti-tumor immune responses were examined in vaccinated animals. D2SC/1–p53M234I and D2SC/1–p53C132F/E168G cells induced strong therapeutic and protective MethA tumor immunity upon transplantation in Balb/c mice. However, in a fraction of immunized mice MethA tumor growth resumed after an extended latency period. Analysis of these tumors indicated loss of p53 expression. Mice, pre-treated with fusion hybrids generated from D2SC/1 and MethA tumor cells, suppressed MethA tumor growth and averted adaptive immune escape. Polyclonal B-cell responses directed against various MethA tumor proteins could be detected in the sera of D2SC/1–MethA inoculated mice. Athymic nude mice and Balb/c mice depleted of CD4+ or CD8+ T-cells were not protected against MethA tumor cell growth after immunization with D2SC/1–MethA hybrids. Our results highlight a potential drawback of cancer immunotherapy by demonstrating that the induction of a specific anti-tumor response favors the acquisition of tumor phenotypes promoting immune evasion. In contrast, the application of DC/tumor cell fusion hybrids prevents adaptive immune escape by a T-cell dependent mechanism and provides a simple strategy for personalized anti-cancer treatment without the need of selectively priming the host immune system.

Abstract 2810: Dendritic cell-based immunotherapy in combination with programmed cell death 1 blockade reduced established tumors by activating Th-1 type immune responses in a murine hepatocellular carcinoma model

Abstract Progress in treatments for hepatocellular carcinoma (HCC) has improved the prognosis of patients with HCC. However, HCC is usually associated with cirrhosis and often recurs even after complete treatment of the tumors in the remaining part of the cirrhotic liver. Thus, there is a strong need for the development of a new intervention therapy that suppresses the occurrence or recurrence effectively with fewer side effects. Immunotherapy may be such a treatment and several clinical trials have been performed for HCC treatment. Dendritic cells (DCs) are known as professional antigen-presenting cells characterized by their potent ability to elicit immune responses to tumor-specific T cells against tumor-associated antigens. Programmed cell death 1 (PD-1) has been identified as a marker of exhausted T cells in chronic disease states, and blockade of PD-1-PD-ligand interactions has been shown to partially restore T cell function. In this study, we evaluated the efficacy of the combination of DC-based vaccine and PD-1 blockade, and investigated the mechanisms of the antitumor effects of the combined therapy. In protection model, mice were injected with DCs or/and PD-1-antibody before the murine HCC tumor cell (BNL cell) challenge. 40% of mice treated with both DCs and PD-1-antibody rejected tumor challenge, whereas other groups observed a palpable tumor in all mice tested. In therapeutic model, tumor-bearing mice were inoculated with DCs or/and PD-1-antibody. Significant suppression of outgrowth of the established tumors was observed in the DCs and anti-PD-1 combination treatment group (DCs + anti-PD-1, 138.00 ± 56.66 mm2 vs control, 402.33 ± 40.63 mm2 on day 42, P = 0.0073 vs controls). Immunohistochemical analyses of therapeutic model showed marked infiltration of CD4+ cells, CD8+ cells and CD11c+ cells in the established BNL tumors of mice treated with both DCs and PD-1-antibody. To investigate induction of tumor-specific immune responses, we stimulated splenocytes of DCs or/and PD-1-antibody treated mice twice weekly by DCs in vitro. Significant tumor-specific cytolysis was detected when splenocytes of mice treated with both DCs and anti-PD-1 were used as effector cells (30.0% ± 2.8% for BNL and 7.3% ± 0.1% for YAC-1, effector to target ratio; E/T=40). Our findings suggest that blockade of the PD-1 PD-ligand enhanced the Th1-type antitumor immune responses induced by DC vaccine. The combination of DC vaccine and PD-1 blockade may be a possible candidate for a cancer vaccine for clinical trials. Citation Format: Eiichi Hayashi, Junichi Eguchi, Masashi Sakaki, Hiroyoshi Doi, Risa Oomori, Atsushi Kajiwara, Hitoshi Yoshida, Shigeaki Ishii, Kazumasa Hiroishi, Michio Imawari. Dendritic cell-based immunotherapy in combination with programmed cell death 1 blockade reduced established tumors by activating Th-1 type immune responses in a murine hepatocellular carcinoma model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2810. doi:10.1158/1538-7445.AM2014-2810

New generation dendritic cell vaccine for immunotherapy of acute myeloid leukemia.

Dendritic cell (DC)-based immunotherapy is a promising strategy for the elimination of minimal residual disease in patients with acute myeloid leukemia (AML). Particularly, patients with a high risk of relapse who are not eligible for hematopoietic stem cell transplantation could benefit from such a therapeutic approach. Here, we review our extensive studies on the development of a protocol for the generation of DCs with improved immunogenicity and optimized for the use in cell-based immunotherapy. This new generation DC vaccine combines the production of DCs in only 3 days with Toll-like receptor-signaling-induced cell maturation. These mature DCs are then loaded with RNA encoding the leukemia-associated antigens Wilm's tumor protein 1 and preferentially expressed antigen in melanoma in order to stimulate an AML-specific T-cell-based immune response. In vitro as well as in vivo studies demonstrated the enhanced capacity of these improved DCs for the induction of tumor-specific immune responses. Finally, a proof-of-concept Phase I/II clinical trial is discussed for post-remission AML patients with high risk for disease relapse.

The Need for Improvement of the Treatment of Advanced and Metastatic Cervical Cancer, the Rationale for Combined Chemo-Immunotherapy

The prognosis of patients with metastatic cervical cancer is poor with a median survival of 8-13 months. Despite the potency of chemotherapeutic drugs, this treatment is rarely curative and should be considered palliative only. The last decades, targeted therapies such as immunotherapy have emerged as an attractive option for the treatment of these patients. Immunotherapy can consist of different modalities such as monoclonal antibodies, adoptive lymphocyte transfer and vaccines, which all are intended to augment the antitumor immune responses in cancer patients. The available evidence indicates that both active and adoptive immunotherapeutical strategies are quite effective against small tumor burdens, but are usually insufficient to eradicate the disease in patients with advanced stages of different kinds of cancer, despite strong induction of tumor-specific immune responses. Although chemotherapy and immunotherapy have not shown to be curative as single modalities, accumulating evidence suggests that combinations of these treatments hold potential for improved clinical outcomes in advanced stages of cancer. Therefore, the combination of chemotherapy and immunotherapy is no longer considered incompatible, because of the emerging insight that certain chemotherapy-based cancer treatments may activate the immune system against the tumor through several molecular and cellular mechanisms. Chemotherapeutic agents and immunotherapy may thus be synergistic and enhance the clinical response. In this review, we show the rationale for combined chemo-immunotherapeutic strategies, and summarize recent data from clinical trials performed in patients with different types of cancer. Challenges such as the selection of the optimal dose and treatment schedule, will be discussed as well as the identification of immune-specific biomarkers. Furthermore, we evaluated the long-term clinical outcomes of patients with advanced cervical cancer treated with HPV16 E6/E7 SLP vaccination with or without chemotherapy. Finally, the future of vaccination therapy in combination with chemotherapy for the treatment of cervical cancer is discussed.

Combination Therapy with Local Radiofrequency Ablation and Systemic Vaccine Enhances Antitumor Immunity and Mediates Local and Distal Tumor Regression

PurposeRadiofrequency ablation (RFA) is a minimally invasive energy delivery technique increasingly used for focal therapy to eradicate localized disease. RFA-induced tumor-cell necrosis generates an immunogenic source of tumor antigens known to induce antitumor immune responses. However, RFA-induced antitumor immunity is insufficient to control metastatic progression. We sought to characterize (a) the role of RFA dose on immunogenic modulation of tumor and generation of immune responses and (b) the potential synergy between vaccine immunotherapy and RFA aimed at local tumor control and decreased systemic progression.Experimental DesignMurine colon carcinoma cells expressing the tumor-associated (TAA) carcinoembryonic antigen (CEA) (MC38-CEA+) were studied to examine the effect of sublethal hyperthermia in vitro on the cells’ phenotype and sensitivity to CTL-mediated killing. The effect of RFA dose was investigated in vivo impacting (a) the phenotype and growth of MC38-CEA+ tumors and (b) the induction of tumor-specific immune responses. Finally, the molecular signature was evaluated as well as the potential synergy between RFA and poxviral vaccines expressing CEA and a TRIad of COstimulatory Molecules (CEA/TRICOM).Results In vitro, sublethal hyperthermia of MC38-CEA+ cells (a) increased cell-surface expression of CEA, Fas, and MHC class I molecules and (b) rendered tumor cells more susceptible to CTL-mediated lysis. In vivo, RFA induced (a) immunogenic modulation on the surface of tumor cells and (b) increased T-cell responses to CEA and additional TAAs. Combination therapy with RFA and vaccine in CEA-transgenic mice induced a synergistic increase in CD4+ T-cell immune responses to CEA and eradicated both primary CEA+ and distal CEA– s.c. tumors. Sequential administration of low-dose and high-dose RFA with vaccine decreased tumor recurrence compared to RFA alone. These studies suggest a potential clinical benefit in combining RFA with vaccine in cancer patients, and augment support for this novel translational paradigm.

Abstract 2613: Prostate cancer stem/initiating cells are targets of both innate and adaptive immunity and elicit potent immune responses against autochthonous prostate tumors.

Abstract Purpose. Objectives of this study were to investigate if prostate cancer stem/initiating cells (CSC) obtained from autochthonous tumors possess the molecular characteristics that allow their in vitro and in vivo recognition by cells of the innate and/or adaptive arms of the immune system, and if CSC are source of antigens for the induction of tumor-specific immune responses. Material and Methods. CSC lines established from the prostate of transgenic adenocarcinoma of the mouse prostate (TRAMP) mice were assessed in vitro for the expression of tumor-associated antigens and susceptibility to NK and T cell killing. We also investigated NK and T cells immune surveillance against CSC by measuring the frequency of CSC-induced tumors in immunocompetent and selectively immunodeficient mice. Finally, we assessed if vaccination with dendritic cells pulsed with CSC induced a tumor-specific immune response able to delay the growth of both transplantable and autochthonous prostate tumors. Results. CSC expressed prostate cancer associated antigens, MHC I and MHC II molecules and ligands for natural killer (NK) cells. Indeed, CSC were targets of NK and cytotoxic T lymphocytes both in vitro and in vivo. Vaccination with dendritic cells pulsed with apoptotic CSC induced a tumor-specific immune response that delayed tumor growth in mice challenged with prostate CSC, and caused tumor regression in TRAMP mice. Conclusions. CSC are targets of both innate and adaptive immune responses and could be exploited for the design of novel immunotherapeutic approaches against cancer. Citation Format: Elena Jachetti, Stefania Mazzoleni, Matteo Grioni, Alessia Ricupito, Chiara Brambillasca, Luca Generoso, Arianna Calcinotto, Massimo Freschi, Anna Mondino, Rossella Galli, Matteo Bellone. Prostate cancer stem/initiating cells are targets of both innate and adaptive immunity and elicit potent immune responses against autochthonous prostate tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2613. doi:10.1158/1538-7445.AM2013-2613

Clinical Trial of the Intratumoral Administration of Labeled DC Combined With Systemic Chemotherapy for Esophageal Cancer

Esophageal cancer is a highly aggressive disease, and improved modalities for its treatment are needed. We performed chemoimmunotherapy involving the intratumoral administration of 111In-labeled dendritic cells (DC) in combination with preoperative chemotherapy in 5 esophageal cancer patients. Mature DC were generated and traced by scintigraphy after their administration. No adverse events that were directly related to the intratumoral DC administration were observed. Delayed-type hypersensitivity skin tests against keyhole limpet hemocyanin, which was added to the culture medium, detected a positive response in 3 patients, and keyhole limpet hemocyanin antibody production was observed in 4 patients, suggesting that intratumorally administered DC migrate to the lymph nodes, where they function as antigen-presenting cells. However, scintigraphic images obtained after the DC administration demonstrated that the DC remained at the esophageal tumor injection sites in all cases, and no DC accumulation was observed elsewhere. The accumulation of CD83+ cells in the primary tumor was also observed in 2 out of 4 patients in an immunohistochemical analysis using surgically resected specimens. Although the induction of tumor-specific immune responses during chemoimmunotherapy was also analyzed in enzyme-linked immunosorbent assay against 28 tumor antigens, none of the antibodies against the antigens displayed enhanced titers. No changes of NY-ESO-1-specific cellular immune response was observed in a patient who displayed NY-ESO-1 antibody production before the DC administration. These results suggest that the intratumoral administration of 111In-labeled mature DC during chemotherapy does not lead to detectable DC migration from the primary tumor to the draining lymph nodes, and therefore, might not achieve an optimal clinical response.

Abstract 4386: Effects of interferon-α-transduced tumor cell vaccines and blockade of programmed cell death 1 on the growth of established tumors

Abstract Progress in treatments for cancer has improved the prognosis of patients with colorectal cancer. However, there is a strong need for the development of a new intervention therapy that suppresses the occurrence or recurrence effectively with fewer side effects. Immunotherapy may be such a treatment and gene therapy using tumor cells that are genetically modified to produce cytokines has been studied in several therapeutic models. We have previously reported that the interferon (IFN)-α gene-transduced tumor-based vaccination therapy suppresses the outgrowth of established tumors. Although the suppressive effects on established tumors were observed, we did not see reductions in the size of all of the parental tumors. Therefore, further improvements in the treatment are needed before clinical application, we focused on programmed cell death 1 (PD-1), which has been identified as a marker of exhausted T cells. In this study, we evaluated the efficacy of the combination of IFN-γ-transduced tumor cell vaccines and PD-1 blockade, and investigated the mechanisms of the antitumor effects of the combined therapy. A poorly immunogenic murine colorectal cancer cell line, MC38, was transduced to overexpress IFN-γ (MC38-IFNα). In a therapeutic model, parental tumor-bearing mice were inoculated with MC38-IFNα cells and an anti-PD-1 antagonistic antibody. Significant suppression of outgrowth of the established tumors was observed in the IFN-γ and anti-PD-1 combination treatment group (IFN+ anti-PD-1, 174.17 ± 35.54 mm2 vs control, 328.67 ± 26.36 mm2 on day 28, P = 0.0114 vs controls). Immunohistochemical analyses showed marked infiltration of CD4+ cells as well as CD8+ cells in the established tumors of mice treated with both IFN-γ and anti-PD-1. To investigate induction of tumor-specific immune responses, we stimulated splenocytes of IFN-γ or/and anti-PD-1 treated mice twice weekly by DCs in vitro. Significant tumor-specific cytolysis was detected when splenocytes of mice treated with both IFN-γ and anti-PD-1 were used as effector cells (58.1% ± 6.7% for MC38 and 14.1% ± 1.7% for YAC-1, effector:target = 20, P < 0.001). Our findings suggest that blockade of the PD-1 PD-ligand enhanced the Th1-type antitumor immune responses induced by IFN-γ. The combination of IFN-γ gene-transduced tumor cell vaccines and PD-1 blockade may be a possible candidate for a cancer vaccine for clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4386. doi:1538-7445.AM2012-4386

Targeting dendritic cells with antigen via dendritic cell-associated promoters

The induction of tumor-specific immune responses is largely dependent on the ability of dendritic cells (DCs) to present tumor-associated antigens to T lymphocytes. Therefore, we investigated the use of DC-associated promoter-driven genetic vaccines to specifically target DC in vivo. Restricted expression of vaccine-encoding genes in DC should enhance specificity and improves their safety for clinical applications. Hereto, 3-5 kb upstream sequences of the murine genes encoding CD11c, DC-SIGN, DC-STAMP and Langerin were isolated, characterized and subcloned into enhanced green fluorescent protein (EGFP) reporter constructs. Upon electroporation, EGFP was expressed in DC cell lines, but not in other cell lines, confirming DC-restricted promoter activity. When these promoters were cloned into a construct upstream of the gene for ovalbumin (OVA), it appeared that DC-STAMP promoter-driven expression of OVA (pDCSTAMP/OVA) in DC yielded the most efficient OVA-specific CD4+ and CD8+ T-cell responses in vitro. Administration of pDC-STAMP/OVA in vivo, using the tattoo gun vaccination system, evoked specific immune responses as evidenced in a mouse tumor model. Adoptively transferred pDC-STAMP/OVA-transfected DCs induced strong CD8+ T-cell proliferation in vivo. These experiments demonstrate that our DC-directed promoter constructs are potential tools to restrict antigen expression in DC and could be implemented to modulate DC function by the introduction of relevant proteins.

Inflammatory Mediators Hold the Key to Dendritic Cell Suppression and Tumor Progression

Cancer has long been considered a disease that is associated with immune tolerance. Its connection with inflammation initially appears paradoxical. During the last decade, it has become increasingly clear that immune infiltrates form an integral part of tumor and critically contribute to its development and progression. In the tumor milieu, a variety of inflammatory mediators, such as cytokines (IL- 6, IL-10, VEGF, TGFβ, M-CSF and GM-CSF), chemokines (CCL20 and CXCL8), hormones (prostanoids like PGE2), reactive oxygen species and cellular constituents (gangliosides), are continuously produced. These mediators represent a critical interface between immune and neoplastic compartments. Not only do they continuously support tumor survival and expansion, but suppress the function of immune cells, notably, dendritic cells - the powerful antigen presenting cells that are crucial for induction of tumor-specific immune responses. This review summarizes such a dual role of inflammatory factors and discusses the controversies associated with specific mediators including IL-10, GM-CSF and ROS in tumor and immune modulation. Identifying the inflammatory signature of cancer patients hence represents a critical task for individualized immunotherapy in the future.

Low-dose paclitaxel enhances the anti-tumor efficacy of GM-CSF surface-modified whole-tumor-cell vaccine in mouse model of prostate cancer

Chemotherapy combined with a tumor vaccine is an attractive approach in cancer therapy. This study was designed to investigate the optimal schedule and mechanisms of action of a novel GM-CSF (granulocyte-macrophage colony-stimulating factor) surface-modified tumor-cell vaccine in combination with paclitaxel in the treatment of mouse RM-1 prostate cancer. First, the anti-tumor efficiencies of various dosage of paclitaxel (4, 20, 40mg/kg) in combination with the vaccine in different administration sequences were examined in the mouse RM-1 prostate cancer model. Then, the in vivo and in vitro effects of various dosage of paclitaxel on RM-1 cells, T cells, and DCs (dendritic cells) were evaluated. The results showed that: (a) the GM-CSF-surface-modified tumor-cell vaccine was more potent at inducing the uptake of tumor antigens by DCs than irradiated tumor cells plus free GM-CSF; (b) 4mg/kg paclitaxel combined with the GM-CSF-surface-modified tumor-cell vaccine was the most effective at enhancing tumor regression in RM-1 prostate cancer mice when the vaccine was administrated 2days after paclitaxel; and (c) administration of 4mg/kg paclitaxel followed by the vaccine induced the highest degree of CD8(+) T-cell infiltration in tumor tissue, suggesting that the induction of tumor-specific immune response had occurred. These findings suggested that the GM-CSF-surface-modified tumor-cell vaccine may have potential clinical benefit for patients with prostate cancer when it is combined with paclitaxel. Furthermore, the effect of immunochemotherapy depends on careful selection of paclitaxel dosage and the sequence of paclitaxel/vaccine administration.

A Randomized Trial ofEx vivoCD40L Activation of a Dendritic Cell Vaccine in Colorectal Cancer Patients: Tumor-Specific Immune Responses Are Associated with Improved Survival

To determine whether an autologous dendritic cell (DC) vaccine could induce antitumor immune responses in patients after resection of colorectal cancer metastases and whether these responses could be enhanced by activating DCs with CD40L. Twenty-six patients who had undergone resection of colorectal metastases were treated with intranodal injections of an autologous tumor lysate- and control protein [keyhole limpet hemocyanin (KLH)]-pulsed DC vaccine. Patients were randomized to receive DCs that had been either activated or not activated with CD40L. All patients were followed for a minimum of 5.5 years. Immunization induced an autologous tumor-specific T-cell proliferative or IFNγ enzyme-linked immunospot response in 15 of 24 assessable patients (63%) and a tumor-specific DTH response in 61%. Patients with evidence of a vaccine-induced, tumor-specific T-cell proliferative or IFNγ response 1 week after vaccination had a markedly better recurrence-free survival (RFS) at 5 years (63% versus 18%, P = 0.037) than nonresponders. In contrast, no association was observed between induction of KLH-specific immune responses and RFS. CD40L maturation induced CD86 and CD83 expression on DCs but had no effect on immune responses or RFS. Adjuvant treatment of patients after resection of colorectal metastases with an autologous tumor lysate-pulsed, DC vaccine-induced, tumor-specific immune responses in a high proportion of patients. There was an association between induction of tumor-specific immune responses and RFS. Activation of this DC vaccine with CD40L did not lead to increased immune responses.

Induction of Tumor-specific Immune Response by Gene Transfer of Hsp70-cell-penetrating Peptide Fusion Protein to Tumors in Mice

To induce a tumor-specific immune response by delivering tumor-associated antigens in tumor cells to antigen-presenting cells (APCs), we designed a fusion protein which consists of heat-shock protein 70 (Hsp70) and the C-terminal 34 amino acids of herpes simplex virus VP22 protein (VP22(268-301)), the former having a peptide binding domain and an ability to be recognized by APCs, and the latter able to achieve cell penetration. Hsp70-VP22(268-301), the fusion protein, was efficiently taken up by mouse dendritic cell (DC) line DC2.4. Major histocompatibility complex (MHC) class I-restricted presentation of an epitope peptide of ovalbumin (OVA) was examined in DC2.4, and Hsp70-VP22(268-301) significantly increased the presentation of the peptide compared with Hsp70. Electroporation-assisted injection of naked plasmid vector expressing Hsp70-VP22(268-301) (pHsp70-VP22(268-301)) into subcutaneous tumors of EG7-OVA, a mouse lymphoma-expressing OVA, significantly increased the survival of mice compared with the same treatment with pHSp70, a plasmid expressing Hsp70. Splenocytes from the pHsp70-VP22(268-301)-treated mice exhibited cytolytic activity against both EG7-OVA and the parent EL4, but not against mouse melanoma B16-F10, suggesting that not only OVA-derived antigens but those common to EG7-OVA and EL4 are delivered to APCs. These results provide a new therapeutic method to induce tumor-specific antitumor immunity without identifying nor isolating tumor-associated antigens.

Heat-shock proteins in cancer vaccines: agents of antigen cross-presentation

Heat-shock proteins (HSPs) derived from tumors are capable of eliciting an anticancer immune response by facilitating antigen cross-presentation in antigen-presenting cells (APCs). This process involves the ability of such chaperones to bind tumor antigens and facilitate their uptake by APCs. Recent evidence reveals that HSP–tumor antigen complexes bind cell surface proteins on APCs that mediate complex internalization and antigen-processing events, as well as inducing an innate immune response. Binding of HSPs to surface receptors is, thus, an imposing gateway to the induction of tumor-specific immune responses. Extensive studies in animals have indicated the usefulness of such HSP-based immunotherapy in killing established tumors and causing tumor regression. Currently, one HSP, the endoplasmic reticulum stress-response protein Gp96 is undergoing clinical trials for cancer treatment and has yielded promising results, including the induction of anti-tumor immunity and some benefit for patients when administered as part of a multidose regimen. Future advances in HSP-based immunotherapy will be aided by an understanding of the mechanisms by which HSP–peptide complexes induce innate and adaptive immunity to tumor cells and target the killing of primary and metastatic cancer cells.

Lentiviruses in Cancer Immunotherapy

Lentiviral vectors have emerged as promising tools for cancer immunotherapy owing to their capacity to transduce a wide range of different cell types, including dendritic cells (DCs), the key regulators of immunity. Ex vivo transduced DCs proved to be potent inducers of strong antigen-specific T-cell responses, both in vitro and in vivo. Moreover, lentiviral vectors have been successfully applied for antigen-specific immunization, offering the advantage that the same lentivirus can be used for all patients resulting in an ‘off-the-shelf’ therapeutic. This review provides an update on the state-of-the-art induction of tumor-specific immune responses in vivo upon direct administration of tumor-associated antigen-encoding lentiviruses. Focusing on the cell types transduced, the results of current studies and the explanation for the potency of lentiviral vectors are discussed.