134

Current Cancer Therapy Reviews, 2009, 5, 134-141

Dendritoma Vaccine for Cancer: A Hopeful Approach

Yanzhang Wei1,2,*, Jinhua Li1 and Thomas E. Wagner1,2
1

Oncology Research Institute, Greenville Hospital System University Medical Center, Greenville, SC, USA Department of Biological Sciences, Clemson University, Clemson, SC, USA
Abstract: Cancer vaccines based on dendritic cells (DCs) have been among the most vigorously studied new cancer therapies during the last decade. Although DCs, the most potent antigen-presenting cells in the body, can be loaded with tumor antigens through varying approaches, such as pulsing and gene transfer, DC-tumor fusion represents the most effective way of engaging DCs with tumor antigens. A significant amount of effort has been given to DC-tumor fusion vaccines, yet in most cases the clinical responses have been discouraging. Therefore, further improvement of this promising cancer therapy is urgently required in order to optimize its clinical efficacy. In this review, we briefly summarize the history and current status of DC-tumor fusion vaccines. Then, we focus on discussions of the technology and the clinical applications of the dendritoma vaccine, a highly purified DC-tumor cell vaccine that uniquely presents a tumors entire antigen diversity.

Key Words: Dendritic cells, cancer vaccine, immunotherapy, dendritoma, cell fusion. INTRODUCTION Dendritic cells (DCs) are professional antigen presenting cells that have a vital role in the activation of immune systems. They activate CD4+ T helper cells through their major histocompatibility complex class II (MHC II) pathway, also known as the exogenous antigen processing pathway. They also stimulate CD8+ cytolytic T cells through the MHC I pathway, or the endogenous antigen processing pathway [1]. Recent studies have also shown DCs to be centrally involved in the activation of the innate immune system [2]. Due to these characteristics, DCs have been widely studied as a mediator for cancer immunotherapy [3-5]. Through these studies, different strategies have been developed using DCs to present tumor antigens to the immune system. The first, and perhaps most obvious, strategy is to load the DCs with tumor antigen peptides, proteins, or whole tumor lysates by incubation, a technique called DC pulsing. By using this approach, the induction of antitumor immunity and disease regression have been achieved both in animal and in clinical studies [6-11]. The drawbacks of this approach are that only the exogenous antigen processing pathway of the DCs can be utilized and, therefore, it is probable that only the CD4+ T cells will be activated. An alternative way to load DCs with tumor antigens is to transfer tumor-antigen-encoding genes or mRNA into DCs. A significant effort has been employed using this approach to engage DCs [12-16], however success has been limited due to the fact that only a small number of tumor antigen genes have been identified and can be transferred. Also, gene transfer into DCs can only engage the endogenous antigen processing pathway of DCs and therefore, it is probable that only the CD8 + T cells can be activated. Another potential problem with tumor-antigen gene transfer is the involvement of viral vectors such as retrovirus and adenovirus vectors, which complicate immune responses. Given the disadvantages of the afore-mentioned approaches, an ideal solution would be to get the entire tumor genome into the dendritic cell so that the entire portfolio of tumor antigens would be expressed within the DC. The ideal, and perhaps obvious, approach is to create a hybrid between a dendritic cell and a tumor cell. Furthermore, during the process of hybrid formation, exposing the DCs to tumor cells will allow the DCs to be loaded with tumor antigens through the exogenous antigen processing pathway. In this review, we will briefly summarize the history and current status of DC-tumor fusion vaccines and focus on the dendritoma vaccine, which is a highly purified and uniquely tumordescriptive DC-tumor fusion vaccine. A. FUSION OF DCS WITH TUMOR CELLS As a technique, cell fusion has been used for a long timefrom virus-mediated cell fusion [17], to polyethylene-glycol (PEG) induced cell fusion [18, 19], to electric-pulse induced cell fusion, or electrofusion [20]. Although cell-fusion technology has been used for various purposes [21-28], the most successful story was the production of hybridomas to generate monoclonal antibodies [18, 19]. Further success was achieved when the technology was utilized to generated hybrids from DCs and tumor cells for cancer vaccinations. i. A Brief History of DC-Tumor Fusion The pioneering work of Gong et al. [29] opened a new field of cancer immunotherapy by fusing DCs and tumor cells. Their work fused MUC1 transfected mouse MC38 adenocarcinoma tumor cells with DCs by PEG fusion and demonstrated that the fused cells (FC/MUC1) induced tu 2009 Bentham Science Publishers Ltd.

*Address correspondence to this author at the Oncology Research Institute, 900 W. Faris Road, Greenville, SC 29605, USA; Tel: 864-455-5341; E-mail: ywei@ghs.org 1573-3947/09 $55.00+.00

Dendritoma Vaccine for Cancer: A Hopeful Appraoch

Current Cancer Therapy Reviews, 2009, Vol. 5, No. 2

135

mor-cell specific immune-responses that eliminated established pulmonary metastases. Since then, this research field has expanded quickly and dramatically. This approach has been applied to many tumor models, with encouraging results. These include melanoma [30-35], fibrosarcoma [36, 37], glioma [38, 39], lung carcinoma [40], colon carcinoma [41-43], hepatocellular carcinoma [44, 45], renal cell carcinoma [46], and others. Various methods have been attempted for the fusion of DCs and tumor cells, including chemical [29-31, 36, 38, 39, 41, 42, 45, 47-52], physical [32, 33, 43, 46, 49, 53-58], and biological [34, 35] methods. These studies not only generated some favorable results, but also confirmed the concept that DCtumor fusion, through which tumor antigens can be presented to immune systems by both the exogenous and endogenous antigen processing pathways, is a more powerful approach to induce antitumor immunity than tumor antigen pulsing or tumor-antigen gene transfer [59-65]. Clinical studies using the DC-tumor fusion approach have been tested in many diseases as well, including melanoma [58, 66], malignant glioma [51, 67], breast cancer [68, 69] and renal cell carcinoma [69, 70]. Although some favorable immunological results were observed from these clinical studies, and safety has been confirmed to be a non-issue with this approach, the overall clinical outcomes of these studies have been discouraging. ii. Combination of DC-Tumor Fusion with Other Agents Many approaches have been examined in order to enhance the antitumor clinical-response induced by DC-tumor fusion vaccines in cancer patients. Cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-2 (IL-2), interleukin-12 (IL-12), or interleukin-18 (IL-18) were introduced into the DC-tumor hybrids [30, 36]. Cytokines and/or other agents were also used as adjuvants to enhance the activity of DC-tumor fusion. These include IL-2, IL-12, IL-18 [71-75], Toll-like receptor (TLR) agonists such as CpG oligodeoxynucleotide (ODN) [76, 77], OK432 [78, 79] and others. iii. Unstable Fusion Rate of DC-Tumor Fusion Although fusion rates as high as 50% by PEG [30, 31, 44, 48, 49] and 83.1% by electrofusion [55] have been reported, how to achieve a stable and high DC-tumor cell fusion rate still presents a major challenge to researchers, especially when patients primary tumor cells are used. With our extensive fusion experience, the highest fusion rate of DCs and primary tumor-cells, either by PEG or by electrofusion, has been below 10% (unpublished data). Due to the lack of an effective way to purify the hybrids from the fusion mixture, in most published studies, the vaccines contained very small numbers of fused cells, but are contaminated with large numbers of un-fused tumor cells, un-fused DCs, selfself fused tumor cells, or self-self fused DCs. The injection of this mixture could easily overwhelm the immune system and restrict immune responses from reaching maximum capacity. This argument may represent a good explanation why most favorable results were achieved in animal models but not in human clinical trials.

iv. Hybrid Purification from DC-Tumor Fusion Various methods have been examined to purify the hybrids from DC-tumor fusions. Some researchers have taken advantage of the adherent feature of some tumor cell lines by incubating fusion mixture for a short period of time to allow these cell hybrids to attach to the culture flask, followed by removal of the non-adherent DCs and collection of the loosely-attached fused cells by pipette aspiration, while leaving the tightly attached, unfused tumor cells in the culture flask [5, 29]. Despite the ease of this method with model cell-lines, this approach is not practical for clinical settings because most patient primary tumor-cells are non-adherent cells or there is no adherent difference between adherent tumor-cells and fused hybrids. An alternative method to purify DC-tumor hybrids is to fuse DCs and tumor cells fluorescently labeled with antibodies specific for DC or tumor markers and sort the unique dual colored hybrids by FACS [64]. This approach is fraught with two problems: 1) labeling of DCs with antibodies may compromise their function; and 2) tumor-cell specific markers are not always available, especially for patients primary tumor-cells. Another way to purify fused DC-tumor hybrids involves the genetic labeling of DCs. Phan et al. [34] transfected DCs with a mouse tyrosinase-GFP fusion reporter-gene that is under the control of a mouse melanoma-specific-tyrosinase promoter and fused the transfected DCs with mouse B16 melanoma-tumor cells. As a result, only the hybrid cells expressed GFP, which allowed them to be purified by FACS sorting. Although this approach is very cleverly novel and elegantly marks the hybrids in an extremely accurate manner, it is too complicated and time consuming to transfect a large numbers of patients DCs in a clinical setting. B. DENDRITOMA VACCINE i. PKH Dyes Our group was the first to report a simple and rapid method (dendritoma technology, see below) for purifying the hybrids from a DC-tumor fusion mixture by introducing distinct cyanine PKH fluorescent dyes into the DCs and tumor cells prior to fusion such that the fusion product could be distinguished from tumor and DC cells allowing for instant purification and isolation of the fused DC-tumor cells. The PKH dyes developed by Horan [80-82] permit stable, reproducible cell-labeling through the incorporation of highly aliphatic-reporter molecules containing fluorochrome head groups into the lipid bi-layer of cytoplasmic membranes. Once incorporated into the lipid bilayer, the probes are trapped within the membrane because of their inherent insolubility and the probes remain bound to the cell membranes the life of the cell. The effects of PKH dye labeling on a variety of mammalian cells have been examined. The proliferation of tumorinfiltrating lymphocytes was not affected by labeling with PKH-26GL and no toxic effect was noted in in vivo experiments [83]. Cytotoxic effects were not found in labeling leukemic cell lines or other cells with PKH dyes [84-86]. A cell tracking study in baboons using PKH labeled platelets found no toxic effect [87]. Oh et al. [88] stained several different

136 Current Cancer Therapy Reviews, 2009, Vol. 5, No. 2

Wei et al.

changes were observed. In conclusion, the PKH dyes are safe to use in animals and/or patients [our unpublished data]. FDA approved the use of PKH dyes at the doses mentioned above for clinical trials under IND process. ii. Dendritoma Technology Dendritomas are highly purified fusion hybrids of DCs and tumor cells. Before fusion, DCs are stained with either green fluorescent dye PKH2-GL or PKH67-GL; and tumor cells are stained with the red fluorescent dye PKH26-GL. The green DCs and -ray-irradiated, red tumor cells are mixed and fused by PEG or electrofusion. The duel-colored hybrids or dendritomas in the fusion mixture are separated by FACS sorting. Using this technology, the purity of dendritomas can be as high as 99%. Fig. (1) shows a diagram of dendritoma production. Dendritic Cell Generation. DCs are generated from bone marrow DC precursor cells or monocytes isolated from peripheral blood mononuclear-cells (PBMCs). The bonemarrow precursor cells, or monocytes, are cultured in either complete DC medium (RPMI 1640, 10% serum, 800U/ml GM-CSF, 1000U/ml IL-4, and 100
g/ml gentamicin) or commercial serum-free DC medium such as the CellGro DC medium (CellGenix, Inc.) for 8-10 days. The culture medium is refreshed by replacing half of it with fresh DC medium every 3 days. DCs are matured by adding maturation reagents such LPS or TNF-
24 or 48 hours before harvesting. Tumor Cell Preparation. Although cultured tumor-cell lines can be used to make dendritomas in model studies in laboratory animals, un-cultured, primary tumor cells must be used in clinical applications of DC-tumor cell vaccines. Primary tumor cells are isolated from fresh tumor tissues. Briefly, after separating fat and necrotic tissue away from the tumor tissue, the tumors are cut into small pieces and transferred into cell culture flasks containing tumor-tissue digestion medium (DMEM, 5mM Ca++, 0.5pz collagenase NB6, DNaseI). After rocking for 1-2 hours at 37 C, the cell suspension is collected from the flask and filtered through a 100Hm cell strainer. Red blood cells (RBC) in the tumor cell preparation are removed by ACK lysing. The tumor cells are then characterized by pathological, flow cytometric, and/or immunocytochemical analysis. PKH Dye Staining and Fusion of DCs and Tumor Cells. The DCs are stained green using the PKH2-GL or PKH67-GL fluorescent dyes and the tumor cells are stained red using the PKH26-GL fluorescent dye as outlined by the protocol from the vendor (Sigma, Inc.). Immediately before fusion, the red tumor cells are -irradiated with a single dose of 5000 rads. The green dendritic cells are then mixed with the red tumor cells at different ratios from 10:1 to 1:1. For PEG fusion, the mixed cells are suspended in serum-free RPMI 1640 medium, transferred into a 50-ml conical centrifuge tube, and peletted. Cell fusion is performed at 37 oC by placing the tube containing the mixed-cell pellet in a 37oC water bath. One milliliter of the prewarmed 50% PEG/ DMSO is added to the mixed-cell pellet drop-by-drop over one minute, stirring after each drop. After stirring for one more minute, two milliliter pre-warmed RPMI 1640/Hepes is added to the cell mixture drop-by-drop over two minutes, stirring after each drop. With a 10-ml pipette, 7 ml of pre-

Fig. (1). Dendritoma Production.

human cell types with the PKH dyes and observed no effects upon the growth or viability of these cells in vitro . To further evaluate any potentially toxic effect of PKH dyes in patients, we performed a specific toxicological study using higher doses of irradiated tumor cells stained with both cyanine dyes PKH-2GL and PKH-26GL in mice. In this study, one million dye-stained irradiated B16F0 (ATCC# CRL-6322) melanoma cells were injected intravenously into twenty 8-week old female C57BL/6J mice. The mice were monitored twice daily for four weeks and no abnormal behavior was observed. Each animal responded normally to stimulus; no significant differences were observed in the amount of food or water consumed and periods of sleep or activity observed were comparable to control animals which received one million un-stained B16F0 tumor cells each. Urination and defecation were normal and the mice were observed to be identical to control mice in every way during this period. At Day 28, after tumor-cell injection, the mice were sacrificed for gross anatomical analysis. In addition, the tissues of five animals, chosen at random, were sectioned and prepared for histological analysis. Histological sections of the mice studied revealed no significant abnormalities in any organ system. Neither neoplastic nor inflammatory

Dendritoma Vaccine for Cancer: A Hopeful Appraoch

Current Cancer Therapy Reviews, 2009, Vol. 5, No. 2

137

Table 1. Summary of Human Dendritoma Production

Yield Tumor cells 48x106/gram* (8.7x10 to 127x10 ) Dendritic cells 0.8x106/ml apheresis (0.27x10 to 1.7x10 ) Dendritomas PEG fusion Electrofusion 5% (2% to 8%) 3% (1% to 6%) 80% (61% to 95%) 70% (60% to 90%) 8% (2.5% to 13%) 6% (2% to 10%) >90%** >90%**
6 6 6 6

Viability 78% (66% to 88%)

Fusion Rate N/A

Purity N/A

90% (86% to 98%)

N/A

N/A

*Melanoma; ** Depending on the instrumental parameter setting.

warmed RPMI 1640/Hepes is added into the tube drop-bydrop over 2 to 3 minutes. The fusions are then pelleted, resuspended in complete DC medium, and incubated overnight in a humidified 37o C, 5% CO2 incubator. For electrofusion, the green dendritic cell and red tumor cell mixture is washed at least two times with electrofusion medium (0.3 M D-glucose, 18 mM MgCl2, 18 mM CaCl2, 1 mM HEPES, pH 7.2) and transferred into electrofusion chambers (0.4 cm cuvette from BTX). The cells are aligned for 40 seconds with 15 V AC, pulsed with a single 30-
s pulse of 480 V DC, then re-exposed to 15 V AC for 9 seconds to promote post-fusion adherence using the ECM 2001 Electro Cell Manipulator from BTX. The electrically fused cells are then resuspended in complete DC medium and incubated overnight in a humidified 37o C, 5% CO2 incubator. Cell Sorting by FACS. After overnight incubation, the fusion mixture is harvested from the flask; the cells are washed once with PBS and then resuspended in PBS. Cells are then FACS sorted on an instrument such as the Vantage SE (Becton Dickinson) after FACS analysis. Duel colored cells are gated, sorted, and collected. The hybrid cells, or dendritomas, are characterized for purity, viability, and other quality control (QC) parameters. After -irradiation at a dose of 10,000 rads, dendritomas are either directly introduced into experimental animals or patients or cryopreserved for later use. It is more complicated making human autologous dendritomas than making mouse dendritomas or using human tumor cell-lines. The most challenging issue is to generate a sufficient number of viable and sterile tumor cells from dissected human tumor-tissues. Table 1 summarizes the information of human dendritoma production. iii. Dendritomas are Superior Activators of Antitumor Immunity Using this novel technology, dendritomas were produced and compared with fusion mixtures in activating antitumor immunity both in animal and in vitro human studies [89, 90]. In the animal study, we made dendritomas from mouse dendritic cells and B16F0 mouse melanoma tumor cells and performed a series of experiments, including CTL assays, interferon- (IFN-) production by T cells after dendritoma activation, and pulmonary metastasis assays, to compare the antitumor immunities induced by dendritomas or by DC/tumor fusion mixtures. The results clearly demonstrated

that dendritomas, highly purified hybrids of DC-tumor fusion, induced stronger antitumor activity than DC-tumor fusion mixture did [89]. In the in vitro human study, dendritomas were generated using DCs cultured from a patients peripheral blood and primary melanoma tumor cells from the same patient and used to activate T cells also from the same patient. The results confirmed the observation from the animal study: T cells activated by autologous dendritomas differentiated to autologous tumor-cell-specific CTLs and showed enhanced CTL activity when compared with T cells activated by autologous DC-tumor fusion mixtures [90]. iv. Clinical Studies with Dendritomas We have conducted several Phase I and II clinical trials to evaluate the safety and activity of dendritoma vaccines combined with either IL-2 or BCG in cancer patients with melanoma, renal cell carcinoma, and neuroblastoma. In the phase I melanoma clinical trial, ten metastatic melanoma patients all with stage IV diseases were enrolled, and dendritomas were generated using autologous DCs and tumor cells. The initial vaccine was given subcutaneously and followed by IL-2 therapy in serially elevated doses from 3 to 9 million units/m2 for 5 days. Repeated vaccinations were administered without IL-2, at 3 month intervals for a maximum of five times. Immune reactions were measured by monitoring the increase in IFN-  expressing T cells. Vaccine doses ranged from 250,000 to 1,000,000 dendritomas. There was no grade 2 or higher toxicity directly attributable to the vaccine. Eight of nine eligible patients demonstrated immunologic reactions by showing elevated IFN-
expressing T cells. One patient developed a partial response at twelve weeks after the first vaccine. Nine months later, this patient achieved a complete response. This patient is still tumor free at the time of this manuscript writing, almost six years after receiving the first vaccine. It is worth to mention that the purity of dendritomas made from and used for this patient is 90%. In addition, two patients maintained a stable disease state for 9 and 4 months, respectively and one patient had a mixed response [91]. In the Phase I renal-cell carcinoma trial, ten stage IV renal-cell carcinoma patients were studied. Dendritomas were made from autologous DCs and tumor cells and administered by subcutaneous injection. After initial vaccination, three escalating doses of IL-2 (3, 6, and 9 million units each) were administered within five days. This treatment regimen was tolerated well without severe adverse events directly related

138 Current Cancer Therapy Reviews, 2009, Vol. 5, No. 2

Wei et al.

to the dendritoma vaccine. Again, tumor-cell specific immune reactions were measured by the increase in IFN-
expressing T cells. Nine of nine patients eligible for the analysis showed an increase of IFN-
expressing CD4+ T cells after vaccination(s), while five of eight patients eligible for the analysis showed an increase of IFN-
expressing CD8+ T cells. Clinical responses were documented in 40% of the patients: three with stabilization of disease and one with a partial response documented by reduction in tumor size [92]. In the Phase II melanoma clinical trial [93], dendritomas were manufactured from and injected into 15 stage IV melanoma patients followed by a low dose interleukin-2 regime after the first dendritoma vaccine (3mIU/m2/day on days 1, 3, and 5). The patients received various numbers of vaccines (1 to 6) at six-week intervals. Clinical responses, defined as: CR, complete response; PR, partial response; SD, stable disease; MR, mixed response; PD, progressive disease; and NED, no evidence of disease, were monitored. Survival of the patients was monitored as well. Forteen of the fifteen patients were eligible for evaluation. Twelve weeks after the first vaccine, three patients were NED; four patients were SD; two patients were PD/MR; and six patients were PD. By the end of March, 2008, the median survival of patients was 793 days and the average survival was 930 days. Median disease-free progression was 162 days. All patients in this trial survived more than six months, 12 of the 14 (86%) survived more than one year, half of the patients (50%) survived more than two years and five of the patients (36%) have survived longer than three years. Two of the four remaining live patients at the time of this writing are still disease free and have been alive for 3.3 and 4.9 years after the first vaccine injection. Dendritic-cell-mediated cancer vaccination has attracted significant attention in the search of a cancer vaccine as a therapy [94, 95]. However, the application of this therapy in patients has been hindered by the immune tolerance rendered by the CD25+CD4+ suppressor T cells [96]. In order to generate effective antitumor T cell responses, it is necessary to develop vaccines capable of reversing tumor antigen-specific T cell tolerance. Recent studies have demonstrated that the engagement of Toll-like receptors (TLR) on dendritic cells is able to break the T cell tolerance [97, 98]. In an animal study, Yang et al. showed that co-administration of LPS or CpG with dendritic cells loaded with tumor-antigens generated strong antitumor immune response. However, these chemicals cannot be used in patients because of their toxicity. The Mycobacterium bovis bacillus Calmette-Guerin (BCG) has been used as a vaccine for tuberculosis since 1921 [99]. Although the effect of BCG as tuberculosis vaccine is controversial, BCG has been successfully used as a treatment for bladder cancer [100] or as an adjuvant for cancer vaccines [101, 102]. The exact mechanism of action is unknown, but the anti-tumor effect appears to be Tlymphocyte-dependent. We treated a patient who had neuroblastoma with dendritomas made from her own monocytes and tumor cells combined with an adjuvant BCG treatment. The patient had stage

IV disease and had no alternative treatment options available when she was enrolled in the trial. The patient exhibited paraplegia secondary to the tumor around the spinal cord and the primary tumor was suprarenal with erosion through the spinal column. The cancer had spread to distant sites, including the patients bone marrow, liver, and lumbar vertegrae. The bulk tumor from the right suprarental area was surgically removed and used to make dendritomas. The paient received five dendritoma vaccines with approximately 270,000 cells each and five BCG injections (1,000,000 CFU/injection) at the same time the dendritomas were delivered. The patient tolerated the treatment very well. One month after the first vaccine, the sum of four target tumors shrank by 9% and then stablized. When the patient, then unable to walk, was admitted to the trial her physician estimated her life expectancy to be less than three months. This patient clearly benefited from her dendritoma vaccine therapy. Not only did she survive, but her pain lessened significantly following vaccination, she recovered her ability to walk and was able to return to her profession for a period of time. Unfortuntely, two new tumors developed four months after the first vaccine and continued to progress. The patient expired twelve month later. In an on going Phase II clinical trial, we are treating stage IV melanoma patients with dendritoma vaccine with BCG as adjuvents. The data for this trial are not available yet. v. Future of Dendritoma Vaccine Although the aforementioned small-scale clinical studies of dendritoma vaccine therapy show very encouraging results, including the safety of the vaccines, tumor-cell specific immunological responses, and clinical responses, several obstacles need to be removed before it is ready for large scale clinical use as a new cancer therapy. The technical aspects of the manufacturing process for dendritoma production have not been completely optimized. This optimization should address the following issues: 1) The yield of DCs cultured from patients PBMC derived monocytes needs to be improved from the current 60-70% to a higher level. 2) A more efficient and closed DC culture system and primary tumor-cell isolation system needs to be developed. 3) Better electrofusion instruments with higher fusion rates need to be developed, optimized, and universal electrofusion conditions good for any type of tumor cells need to be established. 4) FACS cell sorters designed for clinical use need to be designed and made available. In spite of these existing deficiencies, dendritoma-therapeutic vaccination seems to hold real promise for patients with a wide range of solid tumors. Since the majority of the patients enrolled in the early DCtumor fusion vaccine trials were stage IV patients whose immune system had been significantly compromised either by the disease itself or by previous treatments, the observed results are encouraging. The ideal patients for such treatment would be stage II or III patients. According to our Phase II melanoma trial, the best group of patients would be those with no evidence of disease (NED) after target tumor removal. Finally, more work will need to be done in combining dendritoma vaccines with adjuvants, such as BCG, to

Dendritoma Vaccine for Cancer: A Hopeful Appraoch

Current Cancer Therapy Reviews, 2009, Vol. 5, No. 2

139

engage the innate immune system in this tumor immunotherapy. CONCLUSION Although many laboratory studies and clinical trials have been done in the last decade, DC-tumor fusion vaccine immunotherapy for cancer treatment is still a young field. The success of this novel therapy requires more knowledge about tumor biology, dendritic-cell biology and immunology as well as new techniques for cell preparation and functional analysis. We need to understand, for example, why most of the animal studies demonstrated more favorable results than human clinical studies. With time, immunological knowledge, and additional studies, DC-tumor fusion vaccine, or dendritoma vaccine immunotherapy, can certainly become one of the most effective armaments in the battle against cancer. ACKNOWLEDGEMENTS The authors would like to thank Jillian Danson for her help in editing this review. The clinical trials were funded by the Greenville Hospital System University Medical Center Oncology Research Foundation. REFERENCES
[1] [2] [3] [4] [5] [6] [7] [8] Steinman R. The dendritic cell system and its role in immunogenicity. Annu Rev Immunol 1991; 9: 271-96. Leon B, Ardavin C. Monocyte-derived dendritic cells in innate and adaptive immunity. Immunol Cell Biol 2008; 86: 320-4. Steinman RM. Dendritic cells and the control of immunity: enhancing the efficiency of antigen presentation. Mt Sinai J Med 2001; 68: 106-66. Dauer M, Schnurr M, Eigler A. Dendritic cell-based cancer vaccination: quo vadis? Expert Rev Vaccines 2008; 7: 1041-53. Gong J, Koido S, Calderwood SK. Cell fusion: from hybridoma to dendritic cell-based vaccine. Expert Rev Vaccines 2008; 7: 105568. Celluzzi CM, Mayordomo JI, Storkus WJ, Lotze MT, Falo LD, Jr. Peptide-pulsed dendritic cells induce antigen-specific CTLmediated protective tumor immunity. J Exp Med 1996; 183: 283-7. Mayordomo JI, Zorina T, Storkus WJ, et al. Bone marrow-derived dendritic cells pulsed with synthetic peptides elicit protective and therapeutic antitumor immunity. Nat Med 1995; 1: 1297-302. Paglia P, Chiodoni C, Rodolfo M, Colombo MP. Murine dendritic cells loaded in vitro with soluble protein prime cytotoxic T lymphocytes against tumor antigen in vivo. J Exp Med 1996; 183: 31722. Nestle FO, Aligagic S, Gilliet M, et al. Vaccination of melanoma patients with peptide- or tumor lysate-pulsed dendritic cells. Nat Med 1998; 4: 328-32. Hsu FJ, Benike C, Fagnoni F, et al. Vaccination of patients with Bcell lymphoma using autologous antigen-pulsed dendritic cells. Nat Med 1996; 2: 52-8. Loveland BE, Zhao A, White S, et al. Mannan-MUC1-pulsed dendritic cell immunotherapy: a Phase I trial in patients with adenocarcinoma. Clin Cancer Res 2006; 12: 869-77. Gong J, Chen L, Chen D, et al. Induction of antigen-specific antitumor immunity with adenovirus-transduced dendritic cells. Gene Ther 1997; 4: 1023-8. Tang CK, Lodding J, Minigo G, et al. Mannan-mediated gene delivery for cancer immunotherapy. Immunology 2007; 120: 32535. Koido S, Kashiwaba M, Chen D, et al. Induction of antitumor immunity by vaccination of dendritic cells transfected with MUC1 RNA. J Immunol 2000; 165: 5713-9. Specht JM, Wang G, Do MT, et al. Dendritic cells retrovirally transduced with a model antigen gene are therapeutically effective

[16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28]

[29] [30]

[31]

[32] [33] [34] [35]

[9] [10] [11] [12] [13] [14] [15]

[36]

[37] [38] [39]

against established pulmonary metastases. J Exp Med 1997; 186: 1213-21. Codon C, Watkins SC, Culluzzi CM, Thompson K, Falo LD, Jr. DNA-based immunization by in vitro transfection of dendritic cells. Nat Med 1996; 2: 1122-8. Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 1975; 256: 495-7. Galfre G, Howe SC, Milstein C, Butcher GW, Howard JC. Antibodies to major histocompatibility antigens produced by hybrid cell lines. Nature 1977; 266: 550-2. Margulies DH. Monoclonal antibodies: producing magic bullets by somatic cell hybridization. J Immunol 2005; 174: 2451-2. Shu S, Cochran AJ, Huang RR, Morton DL, Maecker HT. Immune responses in the draining lymph nodes against cancer: implications for immunotherapy. Cancer Metastasis Rev 2006; 25: 233-42. Okada Y, Tadokoro J. The distribution of cell fusion capacity among several cell strains or cells caused by HVJ. Exp Cell Res 1963; 32: 417-30. Okada Y, Yamada K, Tadokoro J. Effect of antiserum on the cell fusion reaction caused by HVJ. Virology 1964; 22: 397-409. Okada Y, Kim J, Maeda Y, Koseki I. Specific movement of cell membranes fused with HVJ (Sendai virus). Proc Natl Acad Sci 1974; 71: 2043-7. Furusawa M, Nishimura T, Yamaizumi M, Okada Y. Injection of foreign substances into single cells by cell fusion. Nature 1974; 249: 449-50. Frye LD, Edidin M. the rapid intermixing of cell surface antigens after formation of mouse-human heterokaryons. J Cell Sci 1970; 7: 319-35. Singer SJ, Nicolson GL. The fluid mosaic model of the structure of cell membranes. Science 1972; 175: 720-31. Rodriguez-Tome P, Lijnzaad P. The radiation hybrid database. Nucleic Acids Res 1999; 27: 115-8. Islam MQ, Meirelles Lda S, Nardi NB, Magnusson P, Islam K. Polyethylene glycol-mediated fusion between primary mouse mesenchymal stem cells and mouse fibroblasts generates hybrid cells with increased proliferation and altered differentiation. Stem Cells Dev 2006; 15-905-19. Gong J, Chen D, Kashiwaba M, Kufe D. Induction of antitumor activity by immunization with fusions of dendritic and carcinoma cells. Nat Med 1997; 3: 558-61. Cao X, Zhang W, Wang J, et al. Therapy of established tumor with a hybrid cellular vaccine generated by using granulocytemacrophage colony-stimulating factor genetically modified dendritic cells. Immunology 1999; 97: 616-25. Wang J, Saffold S, Cao X, Krauss J, Chen W. Eliciting T cell immunity against poorly immunogenic tumors by immunization with dendritic cell-tumor fusion vaccines. J Immunol 1998; 161: 551624. Tanaka H, Shimizu K, Hayashi T, Shu S. Therapeutic immune response induced by electrofusion of dendritic and tumor cells. Cell Immunol 2002; 220: 1-12. Shimizu K, Kuriyama H, Kjaergaard J, et al. Comparative analysis of antigen loading strategies of dendritic cells for tumor immunotherapy. J Immunother 2004; 27: 256-72. Phan V, Errington F, Cheong SC, et al. A new genetic method to generate and isolate small, short-lived but highly potent dendritic cell-tumor cell hybrid vaccines. Nat Med 2003; 9: 1215-9. Hiraoka K, Yamamoto S, Otsuru S, et al. Enhanced tumor-specific long-term immunity of hemagglutinating virus of Japan-mediated dendritic cell-tumor fused cell vaccination by coadministration with CpG oligodeoxynucleotides. J Immunol 2004; 173: 4297-307. Ogawa F, Iinuma H, Okinaga K. Dendritic cell vaccine therapy by immunization with fusion cells of interleukin-2 gene-transduced, spleen-derived dendritic cells and tumor cells. Scand J Immunol 2004; 59: 432-9. Kjaergaard J, Shimizu K, Shu S. Electrofusion of syngeneic dendritic cells and tumor generates potent therapeutic vaccine. Cell Immunol 2003; 225: 65-74. Hayashi T, Tanaka H, Tanaka J, et al. Immunogenicity and therapeutic efficacy of dendritic-tumor hybrid cells generated by electrofusion. Clin Immunol 2002; 104: 14-20. Akasaki Y, Kikuchi T, Homma S, et al. Antitumor effect of immunization with fusions of dendritic and glioma cells in a mouse brain tumor model. J Immunother 2001; 24: 106-13.

140 Current Cancer Therapy Reviews, 2009, Vol. 5, No. 2 [40] [41] [42] [43] Celluzzi CM, Falo LD, Jr. Physical interaction between dendritic cells and tumor cells results in an immunogen that induces protective and therapeutic tumor rejection. J Immunol 1998; 160: 3081-5. Kao JY, Gong Y, Chen CM, Zheng QD, Chen JJ. Tumor-derived TGF-
reduces the efficacy of dendritic cell/tumor fusion vaccine. J Immunol 2003; 170: 3806-11. Takeda A, Homma S, Okamoto T, Kufe D, Ohno T. Immature dendritic cell/tumor cell fusion induce potent antitumor immunity. Eur J Clin Invest 2003; 33: 897-904. Suzuki T, Fukuhara T, Tanaka M, et al. Vaccination of dendritic cells loaded with interleukin-12-secreting cancer cells augments in vivo antitumor immunity: characteristics of syngeneic and allogeneic antigen-presenting cell cancer hybrid cells. Clin Cancer Res 2005; 11: 58-66. Homma S, Toda T, Gong J, Kufe D, Ohno T. Preventive antitumor activity against hepatocellular carcinoma (HCC) induced by immunization with fusions of dendritic cells and HCC cells in mice. J Gastroenterol 2001; 36: 764-71. Zhang JK, Li J, Zhang J, Chen HB. Chen SB. Antitumor immunopreventive and immunotherapeutic effect in mice induced by hybrid vaccine of dendritic cells and hepatocarcinoma in vivo. World J Gastroenterol 2003; 9: 479-84. Siders WM, Vergilis KL, Johnson C, Shields J, Kaplan JM. Induction of specific antitumor immunity in the mouse with the electrofusion product of tumor cells and dendritic cells. Mol Ther 2003; 7: 498-505. Gong J, Koido S, Chen D, et al. Immunization against murine multiple myeloma with fusions of dendritic and plasmacytoma cells is potentiated by interleukin-12. Blood 2002; 99: 2512-7. Liu Y, Zhang W, Chan T, Saxena A, Xiang J. Engineered fusion hybrid vaccine of IL-4 gene-modified myeloma and relative mature dendritic cells enhances antitumor immunity. Leuk Res 2002; 26: 757-63. Lindner M, Schirrmacher V. Tumour cell-dendritic cell fusion for cancer immunotherapy: comparison of therapeutic efficiency of polyethylene-glycol versus electrofusion protocols. Eur J Clin Inves 2002; 32: 207-17. Xia D, Chan T, Xiang J. Dendritic cell/myeloma hybrid vaccine. Methods Mol Med 2005; 113: 225-33. Homma S, Kikuchi T, Ishiji N, et al. Cancer immunotherapy by fusions of dendritic and tumour cells and rh-IL-12. Eur J Clin Invest 2005; 35: 279-86. Kao JY, Zhang M, Chen CM, Chen JJ. Superior efficacy of dendritic cell-tumor fusion vaccine compared with tumor lysate-pulsed dendritic cell vaccine in colon cancer. Immunol Lett 2005; 101: 154-9. Scott-Taylor TH, Pettengell R, Clarke I, et al. Human tumour and dendritic cell hybrids generated by electrofusion: potential for cancer vaccines. Biochem Biophys Act 2000; 1500: 265-79. Jantscheff P, Spagnoli G, Zajac P, Rochlitz CF. Cell fusion: an approach to generating constitutively proliferating human tumor antigen-presenting cells. Cancer Immunol Immunother 2002; 51: 367-75. Goddard RV, Prentice AG, Copplestone JA, Kaminski ER. In vitro dendritic cell-induced T cell responses to B cell chronic lymphocytic leukaemia enhanced by IL-15 and dendritic cell-B-CLL electrofusion hybrids. Clin Exp Immunol 2003; 131: 82-9. Marten A, Renoth S, Heinicke T, et al. Allogeneic dendritic cells fused with tumor cells: preclinical results and outcome of a clinical Phase I/II trial in patients with metastatic renal cell carcinoma. Hum Gene Ther 2003; 14: 483-94. Trevor KT, Cover C, Ruiz YW, et al. Generation of dendritic celltumor cell hybrids by electrofusion for clinical vaccine application. Cancer Immunol Immunother 2004; 53: 705-14. Trefzer U, Herberth G, Wohlan K, et al. Tumor-dendritic hybrid cell vaccination for the treatment of patients with malignant melanoma: immunological effects and clinical results. Vaccine 2005; 23: 2367-73. Steinman RM, Swanson J. The endocytic activity of dendritic cells. J Exp Med 1995; 182: 283-8. Watts C. The exogenous pathway for antigen presentation on major histocompatibility complex class II and CD1 molecules. Nat Immunol 2001; 5: 685-92. Heath WR, Carbone FR. Cross-presentation, dendritic cells, tolerance and immunity. Annu Rev Immunol 2001; 19: 47-64. [62] [63] [64] [65]

Wei et al. Berard F, Blanco P, Davoust J, et al. Cross-priming of nave CD8 T cells against melanoma antigens using dendritic cells loaded with killed allogeneic melanoma cells. J Exp Med 2000; 192: 1535-44. Wolkers MC, Brouwenstijn N, Bakker AH, Toebes M, Schumacher TN. Antigen bias in T cell cross-priming. Science 2004; 304: 13147. Koido S, Ohana M, Liu C, et al. Dendritic cells fusion with human cancer cells: morphology, antigen expression, and T cell stimulation. Clin Immunol 2004; 113: 261-9. Galea-Lauri J, Darling D, Mufti G, Harison P, Farzaneh F. Eliciting cytotoxic T lymphocytes against acute myeloid leukemia-derived antigens: evaluation of dendritic cell-leukemia cell hybrids and other antigen-loading strategies for dendritic cell-based vaccination. Cancer Immunol Immunother 2002; 51: 299-301. Krause SW, Neumann C, Sorturi A, et al. The treatment of patients with disseminated malignant melanoma by vaccination with autologous hybrids of tumor cells and dendritic cells. J Immnunother 2002; 25: 421-8. Kikuchi T, Akasaki Y, Irie M, et al. Results of a Phase I clinical trials of vaccination of glioma patients with fusion of dendritic and glioma cells. Cancer Immunol Immunother 2001; 50: 337-44. Avigan D, Vasir B, Gong J, et al. Fusion cell vaccination of patients with metastatic breast and renal cancer induces immunological and clinical responses. Clin Cancer Res 2004; 101: 4699-708. Avigan DE, Vasir B, George DJ, et al. Phae I/II study of vaccination with electrofused allogeneic dendritic cells/autologou tumorderived cells in patients with stage IV renal cell carcinoma. J Immunother 2007; 30: 749-61. Haenssle HA, Krause SW, Emmert S, et al. Hybrid cell vaccination in metastatic melanoma: clinical and immunologic results of a Phase I/II study. J Immunother 2004; 27: 147-55. Wasir B, Wu Z, Crawford K, et al. Fusions of dendritic cells with breast carcinoma stimulate the expansion of regulatory T cells while concomitant exposure to IL-12, CpG oligodeoxynucleotides, and anti-CD3/CD28 promotes the expansion of activated tumor reactive cells. J Immunol 2008; 181: 808-21. Ladoire S, Arnould L, Apetoh L, et al. Pathologic complete response to neoadjuvant chemotherapy of breast carcinoma is associated with the disappearance of tumor-infiltrating Fox3+ regulatory T cells. Clin Cancer Res 2008; 14: 2413-20. Zou W. Regulatory T cells, tumour immunity and immunotherapy. Nat Rev Immunol 2006; 6: 295-307. Menard C, Martin F, Apetoh L, Bouyer F, Ghiringhelli F. Cancer chemotherapy: not only a direct cytotoxic effect, but also an adjuvant for antitumor immunity. Cancer Immunol Immunother 2008; Iinuma T, Homma S, Noda T, et al. Prevention of gastrointestinal tumors based on adenomatous polyposis coli gene mutation by dendritic cell vaccine. J Clin Invest 2004; 113: 1307-17. Okamota M, Furuichi S, Nishioka Y, et al. Expression of Toll-like receptor 4 on dendritic cells is significant for anticancer effect of dendritic cell-based immunotherapy in combination with an active component of OK-432, a streptococcal preparation. Cancer Res 2004; 64: 5461-70. Heckelsmiller K, Beck S, Rall K, et al. Combined dendritic celland CpG oligonucleotide-based immune therapy cures large murine tumors that resist chemotherapy. Eur J Immunol 2002; 32: 323545. Nakahara S, Tsunoda T, Baba T, Asabe S, Tahara H. Dendritic cells stimulated with bacterial product, OK-432, efficiently induce cytotoxic T lymphocytes specific to tumor rejection peptide. Cancer Res 2003; 63: 4112-8. Yamanaka R, Homma J, Yajima N, et al. Clinical evaluation of dendritic cell vaccination for patients with recurrent glioma: results of a clinical Phase I/II trial. Clin Cancer Res 2005; 11: 4160-7. Melnicoff MJ, Morahan PS, Jensen BD, Breslin EW, Horan PK. In vivo labeling of resident peritoneal macrophages. J Leukoc Biol 1988; 43: 387-97. Horan PK, Slezak SE. Stable cell membrane labeling. Nature 1989; 340: 167-8. Horan PK, Melnicoff MJ, Jensen BD, Slezak SE. Fluorescent cell labeling for in vivo and in vitro cell tracking. Methods Cell Biol 1990; 33: 469-91. Wallace K, Palmer LD, Perry-Lalley D, et al. Mechanisms of adoptive immunotherapy: improved methods for in vitro tracking of tu-

[44]

[66]

[45]

[67] [68] [69]

[46]

[47] [48]

[70] [71]

[49]

[72]

[50] [51] [52]

[73] [74] [75] [76]

[53] [54]

[55]

[77]

[56]

[78]

[57] [58]

[79] [80] [81] [82] [83]

[59] [60] [61]

Dendritoma Vaccine for Cancer: A Hopeful Appraoch mor-infiltrating lymphocytes and lymphokine-activated killer cells. Cancer Res 1993; 53: 2358-67. Kempf VA, Bohn E, Noll A, Bielfeldt C, Autenrieth IB. In vivo tracking and protective properties of Yersinia-specific intestinal T cells. Clin Exp Immunol 1998; 113: 429-37. Ashley DM, Bol SJ, Waugh C, Kannourakis G. A novel approach to the measurement of different in vitro leukaemic cell growth parameters: the use of PKH GL fluorescent probes. Leuk Res 1993; 10: 873-82. Ashley DM, Bol SJ, Waugh C, Kannourakis G. Viable bone marrow stromal cells are required for the in vitro survival of B-cell prescursor acute lymphoblastic leukemic cells. Leuk Res 1995; 2: 113-20. Michelson AD, Barnard MR, Hechtman HB, et al. In vivo tracking of platelets: circulating degranulated platelets rapidly lose surface P-section but continue to circulate and function. Proc Natl Acad Sci USA 1996; 21: 11877-82. Oh DJ, Lee GM, Francis K, Palsson BO. Phototoxicity of the fluorescent membrane dyes PKH2 and PKH26 on the human hematopoietic KKG1a progenitor cell line. Cytometry 1999; 36: 312-8. Li J, Holmes LM, Franek KJ, et al. Purified hybrid cells from dendritic cell and tumor cell fusions are superior activators of antitumor immunity. Cancer Immunol Immnother 2001; 50: 456-62. Holmes LM, Li J, Sticca RP, Wagner TE, Wei Y. A rapid, novel strategy to induce tumor cell-specific cytotoxic T lymphocyte responses using instant dendritomas. J Immunother 2001; 24: 122-9. Wei Y, Sticca RP, Holmes LM, et al. Dendritoma vaccination combined with low dose interleukin-2 in metastatic melanoma patients induced immunological and clinical responses. Int J Oncol 2006; 28: 585-94. Wei Y, Sticca RP, Li J, et al. Combined Treatment of dendritoma vaccine and low dose interleukin-2 in stage IV renal cell carcinoma

Current Cancer Therapy Reviews, 2009, Vol. 5, No. 2

141

[84] [85]

[93] [94] [95] [96]

[86]

[87]

[97] [98] [99] [100] [101] [102]

[88] [89] [90] [91]

[92]

patients induced clinical response: A pilot study. Oncol Rep, 2007; 18: 665-71. Wei Y, Stephenson JJ, Li, J, et al. A phase II clinical study of dendritoma vaccination combined with low dose interleukin-2 in advanced melanoma patients. J Clin Oncol 2007; 25: 130S. Pardoll, DM. Spinning molecular immunology into successful immunotherapy. Nat Rev Immunol 2002; 2: 227-38. Ridgway D. The First 1000 dendritic cell vaccine. Can Invest 2003; 21: 873-86. Oldenhove G, de Heusch M, Urbain-Vansanten G, et al. CD4+ CD25+ regulatory T cells control T helper cell type 1 responses to foreign antigens induced by mature dendritic cells in vivo. J Exp Med 2003; 198: 259-66. Pasare C, Medzhitov R. Toll pathway-dependent blockage of CD4+CD25+ T cell mediated suppression by dendritic cells. Science 2003; 299: 1033-6. Yang Y, Huang CT, Huang X, Pardoll DM. Persistent Toll-like receptor signals are required for reversal of regulatory T cellmediated CD8 tolerance. Nat Immunol 2004; 5: 508-15. Calmette A, Guerin C, Negre L, Boquet A. Premunition des nouveaus-nes contre la tuberculose par le vaccine BCG, 1921-1926. An Inst Pasteur (Paris) 1926; 40: 89-133. Cheng CW, Chan SF, Chan LW, et al. 15-year experience on intravesical therapy of T1G3 urinary bladder cancer: a conservative approach. Jpn J Clin Oncol 2004; 34: 202-5. Sinkovics JG, Horvath JC. Vaccination against human cancer. Int J Oncol 16: 81-96. Volk J, Sel S, Ganser A, Schoffski P. Tumor cell-based vaccination in renal cell carcinoma: rationale, approaches, and recent clinical development. Curr Drug Targets 2002; 3: 401-8.

Received: September 25, 2008

Revised: December 29, 2008

Accepted: February 02, 2009