Comprehensive immunotherapy combined with intratumoral injection of zoledronate-pulsed dendritic cells, intravenous adoptive activated T lymphocyte and gemcitabine in unresectable locally advanced pancreatic carcinoma: a phase I/II trial.

Dendritic cell (DC)-based vaccines prepared using various antigen loading methods have been studied for cancer immunotherapy. The in vivo provocation of immunity by the direct injection of DCs without using tumor-specific antigens into tumors after apoptosis-inducing chemotherapy is more applicable. We previously reported that zoledronate-pulsed DCs (Zol-DCs) may induce tumor-antigen-specific CD8+ T cells by activating Vγ9γδT cells. In this report, we studied the feasibility, safety, and efficacy of a comprehensive immunotherapy involving the combined intratumoral injection of Zol-DC, gemcitabine (GEM) and αßT cells in locally advanced pancreatic carcinoma. Seven of 15 patients showed a stable disease (SD) and most of the patients showed long-term clinical responses. The FACT-BRM score was significantly higher in the patients with SD. Additionally the CD8+/Treg ratio significantly increased in SD patients after treatment. The median over-all survival and progression-free-survival of 15 patients were 12.0 months and 5.5 months, respectively. Patients with a pretreatment neutrophil/lymphocyte ratio (NLR) lower than 5.0 showed significantly longer survival. Even in an analysis limited to the patients with an NLR lower than 5.0, the patients whose CD8+/Treg ratio increased more than twofold tended to survive longer. In conclusion, the comprehensive immunotherapy using Zol-DCs, systemic αßT cells, and GEM may synergistically show a therapeutic effect on locally advanced pancreatic carcinoma. By using appropriate and precise biomarkers, such as NLR and CD8+/Treg ratio, the present comprehensive immunotherapy could be more beneficial for patients with pancreatic carcinoma.

Dendritic cell vaccines loaded with autologous tumor lysates for solid tumors.

Recently, immunotherapy has been utilized as a novel option for the treatment of various malignancies, however, it is necessary to develop immune cell therapy on the basis of the blockade of immune checkpoints, antigen presentation on dendritic cell (DC) vaccines and the high avidity tumor reactive T cells. Autologous tumor lysate-loaded DC vaccines could have a potency to elicit T cells immune response with both epitopes of neoantigen and com- mon antigen; however the innate immunity should be essential for the cross-presentation process. Furthermore, DC vaccines loaded with tumor lysates by electroporation system eli- cited both antigen-specific CD8 and CD4 T cells. In order to break the immunosuppression, the combined therapy of the tumor lysate-loaded DC vaccines and immune checkpoint inhib- itors should be investigated in the future.

Precision cancer immunotherapy: optimizing dendritic cell-based strategies to induce tumor antigen-specific T-cell responses against individual patient tumors.

Most dendritic cell (DC)-based vaccines have loaded the DC with defined antigens, but loading with autologos tumor-derived antigens would generate DCs that activate personalized tumor-specific T-cell responses. We hypothesized that DC matured with an optimized combination of reagents and loaded with tumor-derived antigens using a clinically feasible electroporation strategy would induce potent antitumor immunity. We first studied the effects on DC maturation and antigen presentation of the addition of picibanil (OK432) to a combination of zoledronic acid, tumor necrosis factor-α, and prostaglandin E2. Using DC matured with the optimized combination, we tested 2 clinically feasible sources of autologous antigen for electroloading, total tumor mRNA or total tumor lysate, to determine which stimulated more potent antigen-specific T cells in vitro and activated more potent antitumor immunity in vivo. The combination of tumor necrosis factor-α/prostaglandin E2/zoledronic acid/OK432 generated DC with high expression of maturation markers and antigen-specific T-cell stimulatory function in vitro. Mature DC electroloaded with tumor-derived mRNA [mRNA electroporated dendritic cell (EPDC)] induced greater expansion of antigen-specific T cells in vitro than DC electroloaded with tumor lysate (lysate EPDC). In a therapeutic model of MC38-carcinoembryonic antigen colon cancer-bearing mice, vaccination with mRNA EPDC induced the most efficient anti-carcinoembryonic antigen cellular immune response, which significantly suppressed tumor growth. In conclusion, mature DC electroloaded with tumor-derived mRNA are a potent cancer vaccine, especially useful when specific tumor antigens for vaccination have not been identified, allowing autologous tumor, and if unavailable, allogeneic cell lines to be used as an unbiased source of antigen. Our data support clinical testing of this strategy.

Large-scale expansion of γδ T cells and peptide-specific cytotoxic T cells using zoledronate for adoptive immunotherapy.

Specific cellular immunotherapy for cancer requires efficient generation and expansion of cytotoxic T lymphocytes (CTLs) that recognize tumor-associated antigens. However, it is difficult to isolate and expand functionally active T-cells ex vivo. In this study, we investigated the efficacy of a new method to induce expansion of antigen-specific CTLs for adoptive immunotherapy. We used tumor-associated antigen glypican-3 (GPC3)-derived peptide and cytomegalovirus (CMV)-derived peptide as antigens. Treatment of human peripheral blood mononuclear cells (PBMCs) with zoledronate is a method that enables large-scale γδ T-cell expansion. To induce expansion of γδ T cells and antigen-specific CTLs, the PBMCs of healthy volunteers or patients vaccinated with GPC3 peptide were cultured with both peptide and zoledronate for 14 days. The expansion of γδ T cells and peptide-specific CTLs from a few PBMCs using zoledronate yields cell numbers sufficient for adoptive transfer. The rate of increase of GPC3­specific CTLs was approximately 24- to 170,000-fold. These CD8(+) cells, including CTLs, showed GPC3-specific cytotoxicity against SK-Hep-1/hGPC3 and T2 pulsed with GPC3 peptide, but not against SK-Hep-1/vec and T2 pulsed with human immunodeficiency virus peptide. On the other hand, CD8(-) cells, including γδ T cells, showed cytotoxicity against SK-Hep-1/hGPC3 and SK-Hep-1/vec, but did not show GPC3 specificity. Furthermore, adoptive cell transfer of CD8(+) cells, CD8(-) cells, and total cells after expansion significantly inhibited tumor growth in an NOD/SCID mouse model. This study indicates that simultaneous expansion of γδ T cells and peptide-specific CTLs using zoledronate is useful for adoptive immunotherapy.

Zoledronate-pulsed dendritic cell-based anticancer vaccines.

The addition of zoledronate to tumor-associated antigen (TAA)-loaded dendritic cells (DCs) promotes the activation of interferon γ-secreting Vγ9 γδ T cells, in turn eliciting TAA-specific CD8+ T-cell responses. Immunological responses induced by zoledronate-pulsed DC-based vaccines have been associated with therapeutic effects in clinical trials.

Immunotherapy of autologous tumor lysate-loaded dendritic cell vaccines by a closed-flow electroporation system for solid tumors.

Dendritic cell (DC)-based vaccines with the use of various antigen loading methods have been developed for cancer immunotherapy. Electroporation (EP) of a whole tumor cell lysate into DCs was previously found to be more potent for eliciting antigen-specific CD8 + T-cells compared to co-incubation of tumor cell lysates with DCs in vitro. In the present report, we studied the feasibility, safety and antitumor effect in the clinical use of an EP-DC vaccine for the immunotherapy of various types of human solid tumors. We successfully prepared an autologous tumor lysate-loaded EP-DC vaccine with high cell viability by the closed-flow electroporation system. In the phase I clinical trial, mild adverse events associated with the EP-DC vaccine were found during the treatment of advanced or recurrent cancer, or during the adjuvant therapy of some types of cancer; no autoimmune responses were observed after treatment with the autologous tumor lysate-loaded EP-DC vaccines. For the antitumor effect of the EP-DC vaccine against the 41 various types of solid tumor, the overall response rate [complete remission (CR) + partial response (PR)] was 4.9% (2/41) and the clinical benefit rate [CR+ PR + long stable disease (SD)] was 31.7% (13/41). Furthermore, the delayed-type hypersensitivity (DTH) reactivity was positive in most cases of long SD and the positive rate of DTH was 91.7% (11/12) for the patients with clinical benefit. In conclusion, the safety and feasibility of the EP-DC vaccine with autologous tumor lysates were confirmed, and it was found that the antitumor effect might be associated with the immunological response induced by the EP-DC vaccine for cancer immunotherapy.

Clinical scale electroloading of mature dendritic cells with melanoma whole tumor cell lysate is superior to conventional lysate co-incubation in triggering robust in vitro expansion of functional antigen-specific CTL.

Recent commercial approval of cancer vaccine, demonstrating statistically significant improvement in overall survival of prostate cancer patients has spurred renewed interest in active immunotherapies; specifically, strategies that lead to enhanced biological activity and robust efficacy for dendritic cell vaccines. A simple, widely used approach to generating multivalent cancer vaccines is to load tumor whole cell lysates into dendritic cells (DCs). Current DC vaccine manufacturing processes require co-incubation of tumor lysate antigens with immature DCs and their subsequent maturation. We compared electroloading of tumor cell lysates directly into mature DCs with the traditional method of lysate co-incubation with immature DCs. Electroloaded mature DCs were more potent in vitro, as judged by their ability to elicit significantly (p < 0.05) greater expansion of peptide antigen-specific CD8(+) T cells, than either lysate-electroloaded immature DCs or lysate-co-incubated immature DCs, both of which must be subsequently matured. Expanded CD8(+) T cells were functional as judged by their ability to produce IFN-γ upon antigen-specific re-stimulation. The electroloading technology used herein is an automated, scalable, functionally closed cGMP-compliant manufacturing technology supported by a Master File at CBER, FDA and represents an opportunity for translation of enhanced potency DC vaccines at clinical/commercial scale.

Development of the mucosal vascular system in the distal colon of the fetal mouse.

The formation of the crypt in the distal colon of the mouse was investigated in association with the development of vascular networks. For histological observation, 1-microm cross-sections were made from the distal colon of fetal mice in 13 to 18 days of gestation. Three-dimensional distributions of vascular networks in the organ were observed after perfusing fetuses with rhodamine isothiocyanate-labeled gelatin and immunostaining for laminin to examine the boundary between the epithelium and the mesenchyme. At 13 days of gestation, the distal colon and its epithelium formed a cylindrical tube and a loose primary plexus of vessels was built in the mesenchyme. In the distal colon of 15 days of gestation, the caudal portion began to form the crypt and the vascular plexus built up from a few layers was situated apart from the boundary between the epithelium and the mesenchyme. As the development proceeded, the formation of the crypt occurred in the caudorostral direction. The developing crypt advanced into the vascular plexus, so that a few vessels situated in the mesenchyme between crypts. As the crypt elongated, these vessels formed a small plexus situated perpendicular to the primary plexus, while the primary plexus became monolayered and loosened. The new plexus was composed of ascending vessels and traversing ones, but the regular honeycomb-like plexuses around openings of crypts have not established yet even in 18 days of gestation. The vascular system as well as the crypt in the distal colon will take further a few postnatal weeks to be completed.

Copulsing tumor antigen-pulsed dendritic cells with zoledronate efficiently enhance the expansion of tumor antigen-specific CD8+ T cells via Vgamma9gammadelta T cell activation.

We demonstrate that Vgamma9gammadelta T cells activated by zoledronate can link innate and acquired immunity through crosstalk with dendritic cells (DCs) in a way that can amplify activation and proliferation of tumor antigen-specific CD8+ T cells. DCs pulsed with antigen alone or antigen plus zoledronate were used to stimulate the in vitro expansion of antigen-specific CD8+ T cells. MART-1-modified peptide (A27L peptide) and apoptotic HLA-A*0201-positive, MART-1-positive JCOCB tumor cell lines were used as tumor antigen sources. The percentage of A27L-specific CD8+ T cells within the responding lymphocytes on Day 7 when immature DCs (imDCs) were cultured in the presence of A27L peptide and 0.01 microM zoledronate was significantly higher (P=0.002, n=11) than that observed when imDCs were cultured with the lymphocytes in the presence of the A27L peptide alone. This enhancing effect of zoledronate was significantly reduced when gammadelta T cells were depleted from responding lymphocytes (P=0.030, n=5), indicating that the effect is mediated mainly through Vgamma9gammadelta T cells activated by zoledronate-pulsed imDCs. When imDCs copulsed with zoledronate and apoptotic JCOCB tumor cell lines were used, the percentage of A27L-specific CD8+ T cells was higher than that observed using imDCs with the apoptotic JCOCB lines alone, suggesting that zoledronate treatment of imDCs enhances the cross-presentation ability of DCs. These findings suggest a potentially valuable role for Vgamma9gammadelta T cell activation for expanding antigen-specific CD8+T cells using DCs copulsed with tumor antigen and zoledronate in the design of vaccine therapies for malignancy.

Efficient priming and expansion of antigen-specific CD8+ T cells by a novel cell-based artificial APC.

The ex vivo priming and expansion of human CTL by APC, such as autologous monocyte-derived dendritic cells (DC), has the potential for use in immunotherapy for infectious diseases and cancer. To overcome the difficulty of obtaining sufficient number of autologous DC from patients, we have developed cell-based artificial APC (aAPC), designated Med-APC. These aAPC rapidly activate and expand the corresponding Ag-specific CD8+ T cells when pulsed with CTL epitope peptide(s) as efficiently as mature DC (mDC). We have also shown that Med-APC possess an innate cellular machinery that is sufficient to support the processing of complete Ag into immunodominant peptides, which considerably extends the usefulness of this technology. In addition, we have developed a novel expression vector system that expresses ubiquitinated Ag, resulting in an enhanced APC function of this system. Genetically encoded Ag can be easily introduced into Med-APC by transfection with this vector. Med-APC transfected with ubiquitinated Ag can efficiently expand the corresponding Ag-specific CTL without exogenous peptides. Therefore, Med-APC may have important therapeutic implications for adoptive immunotherapy and can be used for the detection of Ag-specific CTL for immunomonitoring.

The therapeutic potential of immuno-cell therapy of cancer in combination with aminobisphosphonates.

Many university hospitals and cancer centers in Japan have actively investigated the use of autologous activated lymphocyte therapy (ALT). However its therapeutic efficacy was found to be quite limited. The efficacy of aminobisphosphonates (aBPs) has been shown for osteolytic bone disease. aBPs could activate and induce the proliferation of gamma/delta (gamma/delta) T-cells. The application of aBPs to ALT in vivo or ex vivo may be beneficial. A brief overview of aBPs and gamma/delta T-cells is provided, together with some preliminary results and discussion of the therapeutic potential of ALT in combination with aBPs.

Mature dendritic cells are superior to immature dendritic cells in expanding antigen-specific naive and memory CD8+ T cells.

BACKGROUND: For successful dendritic cell (DC)-based immunotherapy, it is critical to identify the most potent stage of human DCs, including immature DCs (imDCs) and mature DCs (mDCs). MATERIALS AND METHODS: imDCs were obtained by culturing monocytes in the presence of GM-CSF and IL-4 for 5- 7 days and imDCs were further cultured for 24-48 h in the presence of TNFalpha, IL-6, IL-1beta and PGE2 to obtain mDCs. Melan-A- and EBV (BRF1) peptides were used and the frequency of antigen-specific CD8+ T cells was assessed using appropriate tetramers. RESULTS: mDCs were potent antigen-presenting cells for the induction and proliferation of antigen-specific naive and memory CD8+ T cells and may overcome regulatory functions that suppress antigen-specific CD8+ T cells. CONCLUSION: Our findings that mDCs can efficiently expand antigen-specific naive and memory CD8 + T cells have important implications in the development of vaccination strategies and support the use of antigen-loaded mature DCs in human clinical trials