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134
Current Cancer Therapy Reviews
, 2009,
5,
134-1411573-3947/09 $55.00+.00 © 2009 Bentham Science Publishers Ltd.
 
Dendritoma Vaccine for Cancer: A Hopeful Approach
Yanzhang Wei
1,2,*
, Jinhua Li
1
and Thomas E. Wagner 
1,2
1
Oncology Research Institute, Greenville Hospital System University Medical Center, Greenville, SC, USA
2
 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 withtumor antigens through varying approaches, such as pulsing and gene transfer, DC-tumor fusion represents the most effec-tive way of engaging DCs with tumor antigens. A significant amount of effort has been given to DC-tumor fusion vac-cines, yet in most cases the clinical responses have been discouraging. Therefore, further improvement of this promisingcancer therapy is urgently required in order to optimize its clinical efficacy. In this review, we briefly summarize the his-tory and current status of DC-tumor fusion vaccines. Then, we focus on discussions of the technology and the clinical ap- plications of the dendritoma vaccine, a highly purified DC-tumor cell vaccine that uniquely presents a tumor’s entire anti-gen diversity.
Key Words:
Dendritic cells, cancer vaccine, immunotherapy, dendritoma, cell fusion.
 
INTRODUCTION
Dendritic cells (DCs) are professional antigen presentingcells that have a vital role in the activation of immune sys-tems. They activate CD4
+
T helper cells through their major histocompatibility complex class II (MHC II) pathway, alsoknown as the exogenous antigen processing pathway. Theyalso 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 involvedin the activation of the innate immune system [2]. Due tothese characteristics, DCs have been widely studied as a me-diator for cancer immunotherapy [3-5].Through these studies, different strategies have been de-veloped using DCs to present tumor antigens to the immunesystem. The first, and perhaps most obvious, strategy is toload the DCs with tumor antigen peptides, proteins, or wholetumor lysates by incubation, a technique called DC pulsing.By using this approach, the induction of antitumor immunityand disease regression have been achieved both in animaland in clinical studies [6-11]. The drawbacks of this ap- proach are that only the exogenous antigen processing path-way of the DCs can be utilized and, therefore, it is probablethat only the CD4
+
T cells will be activated.An alternative way to load DCs with tumor antigens is totransfer tumor-antigen-encoding genes or 
 
mRNA into DCs.A significant effort has been employed using this approachto engage DCs [12-16], however success has been limiteddue to the fact that only a small number of tumor antigengenes have been identified and can be transferred. Also, genetransfer into DCs can only engage the endogenous antigen
*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
  processing pathway of DCs and therefore, it is probable thatonly the CD8
+
T cells can be activated. Another potential problem with tumor-antigen gene transfer is the involvementof viral vectors such as retrovirus and adenovirus vectors,which complicate immune responses.Given the disadvantages of the afore-mentioned ap- proaches, 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 betweena dendritic cell and a tumor cell. Furthermore, during the process of hybrid formation, exposing the DCs to tumor cellswill allow the DCs to be loaded with tumor antigens throughthe 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 vac-cine, which is a highly purified and uniquely tumor-descriptive DC-tumor fusion vaccine.
A. FUSION OF DCS WITH TUMOR CELLS
As a technique, cell fusion has been used for a long time-from virus-mediated cell fusion [17], to polyethylene-glycol(PEG) induced cell fusion [18, 19], to electric-pulse inducedcell fusion, or electrofusion [20]. Although cell-fusion tech-nology has been used for various purposes [21-28], the mostsuccessful story was the production of hybridomas to gener-ate monoclonal antibodies [18, 19]. Further success wasachieved when the technology was utilized to generated hy- brids 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 newfield of cancer immunotherapy by fusing DCs and tumor cells. Their work fused MUC1 transfected mouse MC38adenocarcinoma tumor cells with DCs by PEG fusion anddemonstrated that the fused cells (FC/MUC1) induced tu-
 
 Dendritoma Vaccine for Cancer: A Hopeful Appraoch Current Cancer Therapy Reviews
, 2009,
Vol. 5, No. 2
135
 
mor-cell specific immune-responses that eliminated estab-lished pulmonary metastases.Since then, this research field has expanded quickly anddramatically. This approach has been applied to many tumor models, with encouraging results. These include melanoma[30-35], fibrosarcoma [36, 37], glioma [38, 39], lung carci-noma [40], colon carcinoma [41-43], hepatocellular carci-noma [44, 45], renal cell carcinoma [46], and others. Variousmethods have been attempted for the fusion of DCs and tu-mor 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 somefavorable results, but also confirmed the concept that DC-tumor fusion, through which tumor antigens can be presentedto immune systems by both the exogenous and endogenousantigen processing pathways, is a more powerful approach toinduce antitumor immunity than tumor antigen pulsing or tumor-antigen gene transfer [59-65]. Clinical studies usingthe DC-tumor fusion approach have been tested in manydiseases as well, including melanoma [58, 66], malignantglioma [51, 67], breast cancer [68, 69] and renal cell carci-noma [69, 70]. Although some favorable immunologicalresults were observed from these clinical studies, and safetyhas been confirmed to be a non-issue with this approach, theoverall clinical outcomes of these studies have been discour-aging.
ii. Combination of DC-Tumor Fusion with Other Agents
Many approaches have been examined in order to en-hance the antitumor clinical-response induced by DC-tumor fusion vaccines in cancer patients. Cytokines such as granu-locyte-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 toenhance the activity of DC-tumor fusion. These include IL-2,IL-12, IL-18 [71-75], Toll-like receptor (TLR) agonists suchas 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 re- ported, how to achieve a stable and high DC-tumor cell fu-sion rate still presents a major challenge to researchers, espe-cially when patients’ primary tumor cells are used. With our extensive fusion experience, the highest fusion rate of DCsand 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 mix-ture, in most published studies, the vaccines contained verysmall numbers of fused cells, but are contaminated withlarge numbers of un-fused tumor cells, un-fused DCs, self-self fused tumor cells, or self-self fused DCs. The injectionof this mixture could easily overwhelm the immune systemand restrict immune responses from reaching maximum ca- pacity. This argument may represent a good explanation whymost favorable results were achieved in animal models butnot in human clinical trials.
iv. Hybrid Purification from DC-Tumor Fusion
Various methods have been examined to purify the hy- brids from DC-tumor fusions. Some researchers have takenadvantage of the adherent feature of some tumor cell lines byincubating fusion mixture for a short period of time to allowthese cell hybrids to attach to the culture flask, followed byremoval of the non-adherent DCs and collection of theloosely-attached fused cells by pipette aspiration, while leav-ing the tightly attached, unfused tumor cells in the cultureflask [5, 29]. Despite the ease of this method with modelcell-lines, this approach is not practical for clinical settings because most patient primary tumor-cells are non-adherentcells or there is no adherent difference between adherenttumor-cells and fused hybrids. An alternative method to pu-rify DC-tumor hybrids is to fuse DCs and tumor cells fluo-rescently 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) labelingof DCs with antibodies may compromise their function; and2) tumor-cell specific markers are not always available, es- pecially for patients’ primary tumor-cells.Another way to purify fused DC-tumor hybrids involvesthe genetic labeling of DCs. Phan
et al.
[34] transfected DCswith a mouse tyrosinase-GFP fusion reporter-gene that isunder the control of a mouse melanoma-specific-tyrosinase promoter and fused the transfected DCs with mouse B16melanoma-tumor cells. As a result, only the hybrid cells ex- pressed GFP, which allowed them to be purified by FACSsorting. Although this approach is very cleverly novel andelegantly marks the hybrids in an extremely accurate man-ner, it is too complicated and time consuming to transfect alarge numbers of patients’ DCs in a clinical setting.
B. DENDRITOMA VACCINEi. PKH Dyes
Our group was the first to report a simple and rapidmethod (dendritoma technology, see below) for purifying thehybrids from a DC-tumor fusion mixture by introducing dis-tinct cyanine PKH fluorescent dyes into the DCs and tumor cells prior to fusion such that the fusion product could bedistinguished from tumor and DC cells allowing for instant purification and isolation of the fused DC-tumor cells. ThePKH dyes developed by Horan [80-82] permit stable, repro-ducible cell-labeling through the incorporation of highlyaliphatic-reporter molecules containing fluorochrome headgroups into the lipid bi-layer of cytoplasmic membranes.Once incorporated into the lipid bilayer, the probes aretrapped within the membrane because of their inherent in-solubility and the probes remain bound to the cell mem- branes the life of the cell.The effects of PKH dye labeling on a variety of mammal-ian cells have been examined. The proliferation of tumor-infiltrating lymphocytes was not affected by labeling withPKH-26GL and no toxic effect was noted in
in vivo
experi-ments [83]. Cytotoxic effects were not found in labeling leu-kemic cell lines or other cells with PKH dyes [84-86]. A celltracking study in baboons using PKH labeled platelets foundno toxic effect [87]. Oh
et al.
[88] stained several different
 
136
Current Cancer Therapy Reviews
, 2009,
Vol. 5, No. 2 Wei et al.
human cell types with the PKH dyes and observed no effectsupon the growth or viability of these cells
in vitro
.To further evaluate any potentially toxic effect of PKHdyes in patients, we performed a specific toxicological studyusing higher doses of irradiated tumor cells stained with bothcyanine dyes PKH-2GL and PKH-26GL in mice. In thisstudy, one million dye-stained irradiated B16F0 (ATCC#CRL-6322) melanoma cells were injected intravenously intotwenty 8-week old female C57BL/6J mice. The mice weremonitored twice daily for four weeks and no abnormal be-havior was observed. Each animal responded normally tostimulus; no significant differences were observed in theamount of food or water consumed and periods of sleep or activity observed were comparable to control animals whichreceived one million un-stained B16F0 tumor cells each.Urination and defecation were normal and the mice wereobserved to be identical to control mice in every way duringthis period. At Day 28, after tumor-cell injection, the micewere sacrificed for gross anatomical analysis. In addition, thetissues of five animals, chosen at random, were sectionedand prepared for histological analysis. Histological sectionsof the mice studied revealed no significant abnormalities inany organ system. Neither neoplastic nor inflammatorychanges were observed. In conclusion, the PKH dyes are safeto use in animals and/or patients [our unpublished data].FDA approved the use of PKH dyes at the doses mentionedabove for clinical trials under IND process.
 ii. Dendritoma Technology
Dendritomas are highly purified fusion hybrids of DCsand 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 aremixed and fused by PEG or electrofusion. The duel-coloredhybrids or dendritomas in the fusion mixture are separated by FACS sorting. Using this technology, the purity of den-dritomas 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 bone-marrow precursor cells, or monocytes, are cultured in either complete DC medium (RPMI 1640, 10% serum, 800U/mlGM-CSF, 1000U/ml IL-4, and 100
g/ml gentamicin) or commercial serum-free DC medium such as the CellGro DCmedium (CellGenix, Inc.) for 8-10 days. The culture mediumis refreshed by replacing half of it with fresh DC mediumevery 3 days. DCs are matured by adding maturation rea-gents such LPS or TNF-
24 or 48 hours before harvesting.
Tumor Cell Preparation.
Although cultured tumor-celllines can be used to make dendritomas in model studies inlaboratory animals, un-cultured, primary tumor cells must beused in clinical applications of DC-tumor cell vaccines. Pri-mary tumor cells are isolated from fresh tumor tissues.Briefly, after separating fat and necrotic tissue away from thetumor tissue, the tumors are cut into small pieces and trans-ferred into cell culture flasks containing tumor-tissue diges-tion medium (DMEM, 5mM Ca
++
, 0.5pz collagenase NB6,DNaseI). After rocking for 1-2 hours at 37° C, the cell sus- pension is collected from the flask and filtered through a100
H
m cell strainer. Red blood cells (RBC) in the tumor cell preparation are removed by ACK lysing. The tumor cells arethen characterized by pathological, flow cytometric, and/or immunocytochemical analysis.
PKH Dye Staining and Fusion of DCs and TumorCells.
The DCs are stained green using the PKH2-GL or PKH67-GL fluorescent dyes and the tumor cells are stainedred using the PKH26-GL fluorescent dye as outlined by the protocol from the vendor (Sigma, Inc.). Immediately beforefusion, the red tumor cells are
-irradiated with a single doseof 5000 rads. The green dendritic cells are then mixed withthe red tumor cells at different ratios from 10:1 to 1:1. For PEG fusion, the mixed cells are suspended in serum-freeRPMI 1640 medium, transferred into a 50-ml conical centri-fuge tube, and peletted. Cell fusion is performed at 37
o
C by placing the tube containing the mixed-cell pellet in a 37
o
Cwater 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 onemore minute, two milliliter pre-warmed RPMI 1640/Hepes isadded 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.
 

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