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BioMed Research International
Volume 2013 (2013), Article ID 492372, 14 pages
What Is Recent in Pancreatic Cancer Immunotherapy?
1Department of Internal Medicine, University of Florence and Patologia Medica Unit Department of Biomedicine, Azienda Ospedaliero-Universitaria Careggi, 50134 Florence, Italy
2Department of Medical and Surgical Critical Care, University of Florence and Patologia Medica Unit Department of Biomedicine, Azienda Ospedaliero Universitaria Careggi, 50134 Florence, Italy
3Center of Oncologic Minimally Invasive Surgery, University of Florence, Largo Brambilla 3, 50134 Florence, Italy
4Division of Immunology, Department of Internal Medicine, University of Florence, Viale Pieraccini, 6, 50134 Florence, Italy
Received 21 May 2012; Accepted 6 July 2012
Academic Editor: Julie Curtsinger
Copyright © 2013 Elena Niccolai et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Pancreatic cancer (PC) represents an unresolved therapeutic challenge, due to the poor prognosis and the reduced response to currently available treatments. Pancreatic cancer is the most lethal type of digestive cancers, with a median survival of 4–6 months. Only a small proportion of PC patients is curative by surgical resection, whilst standard chemotherapy for patients in advanced disease generates only modest effects with considerable toxic damages. Thus, new therapeutic approaches, specially specific treatments such as immunotherapy, are needed. In this paper we analyze recent preclinical and clinical efforts towards immunotherapy of pancreatic cancer, including passive and active immunotherapy approaches, designed to target pancreatic-cancer-associated antigens and to elicit an antitumor response in vivo.
Pancreatic cancer (PC) represents an unresolved therapeutic challenge, due to the poor prognosis and the reduced response to currently available treatments. Pancreatic cancer is the most lethal type of digestive cancers, with a median survival (MS) of 4–6 months . There are three principal PC types: ductal adenocarcinoma, neuroendocrine tumors (rare), and cystic neoplasm (less than 1% of pancreatic cancers) . Pancreatic ductal adenocarcinoma accounts for 90% of cancers of the pancreas and has the poorest outcome, representing the 4th most common cause of cancer-related death among men and women .
The only potentially curative therapy for pancreatic cancer is surgical resection. Unfortunately, only 20% PC patients are resectable at the time of diagnosis, and among those patients who undergo resection and have tumor-free margins, the 5-year survival rate after surgery is 10% to 25% . Gemcitabine, with or without erlotinib, represents the standard chemotherapy but the benefit is only modest, and most patients do not survive longer than 6 months [4, 5].
Development of novel agents and approaches is urgently needed in conjunction with improvement in access to clinical trials for patients. Since there are different evidences that pancreatic adenocarcinomas elicit antitumor immune responses [6–9] specific immunotherapy could be of great importance in the PC treatment. In support of the PC-specific immunotherapy approaches there are numerous data showing how PC patients generate B and T cells specific to antigens expressed on autologous pancreatic tumor cells [10–12], such as Wilms’ tumor gene 1 (WT1) (75%) , mucin 1 (MUC1) (over 85%) , human telomerase reverse transcriptase (hTERT) (88%) , mutated K-RAS (73%) , survivin (77%) , carcinoembryonic antigen (CEA) (over 90%) , HER-2/neu (61.2%) , p53 (67%) , and α-enolase . Furthermore, the analysis of immune infiltrates in human tumors has demonstrated a positive correlation between prognosis and presence of humoral response to pancreatic antigens (MUC-1 and mesothelin) [8, 9, 22] or of tumor-infiltrating T cells .
In this paper we analyze recent preclinical and clinical efforts towards immunotherapy of pancreatic cancer, including passive immunotherapy approaches, such as the use of antibodies or effector cells generated in vitro, and active immunotherapic strategies, whose goal is to stimulate an antitumor response in vivo, by means of vaccination.
2. Passive Immunotherapy
2.1. Humoral Immunity: The Role of Monoclonal Antibodies
Specific recognition and elimination of pathological organisms or malignant cells by antibodies were proposed over a century ago by Paul Ehrlich, who is credited for conceptualizing the “magic bullet” theory of targeted therapy. Over the past 30 years, antibody cancer therapeutics have been developed and used clinically in aneffort to realize the potential of targeted therapy. Antibodies can target antigens differentially expressed in tumor cells (tumor-associated antigens (TAAs)) or can be used to block molecules involved in cancer progression or angiogenesis. The immunoglobulins can invoke tumor cell death by blocking ligand-receptor growth and survival pathways. In addition, innate immune effector mechanisms: antibody-dependent cellular cytotoxicity (ADCC), complement-mediated cytotoxicity (CMC), and antibody-dependent cellular phagocytosis (ADCP), are emerging as equally important .
Although unconjugated antibodies have had efficacy, molecular genetics and chemical modifications to monoclonal antibodies (mAbs) have advanced their clinical utility. For example, modification of immune effector engagement has improved pharmacokinetic profiles, and conjugating cytotoxic agents to mAbs has enhanced targeted therapeutic delivery to tumors. The increasing facility of antibody modifications has made it possible to construct diverse and efficacious mAb-based therapeutics.
The humoral immune response to mesothelin has been found to be a favorable prognostic factor for pancreatic cancer [8, 22, 25, 26]. Mesothelin is a 40 kDa protein present in normal mesothelial cells of the pericardium, pleura, and peritoneum, but overexpressed in mesotheliomas ovarian cancers  and detected in 90–100% of pancreatic adenocarcinomas [28, 29]. Different antibodies to mesothelin have been studied and in particular SS1P, a murine single-chain Fv, specific for human mesothelin, which has been fused to PE38, a 38 kDa portion of Pseudomonas exotoxin A (PE-A). After binding to mesothelin and subsequent internalization into cells, it inhibits protein synthesis and results in apoptosis . In phase I clinical studies SS1P was found to be well tolerated, with self-limiting pleuritis as the dose-limiting toxicity. Also, the administration of a version of SS1P with releasable PEGylation resulted in complete regression of a mesothelin-expressing human carcinoma in mice with only a single dose [30–32]. MORAb-009, a monoclonal antibody against mesothelin, is being tested in a phase I trial of 11 patients (three with pancreatic cancer) . One of them who had previously progressed on gemcitabine showed disease stabilization on computed tomography (CT) and a drop in CA19-9 (carbohydrate antigen 19-9). Two fully human, antihuman mesothelin antibodies, M912 and HN1, have been developed, which bind mesothelin-positive cells and result in their lysis via ADCC [34, 35]. Similar to SS1P, HN1 has been fused to truncated PE-A immunotoxin, although its binding site on mesothelin probably binds a distinct but overlapping epitope to that of SS1P .
MUC1 (mucin-1, CD227) is a polymorphic, glycosylated type I transmembrane protein present in glandular epithelium of different tissues (pancreas, breast, lung) and overexpressed (aberrantly glycosylated) in 90% of pancreatic cancers [36, 37]. It inhibits cell-cell and cell-stroma interactions and functions as a signal transducer in the cancer progression, including tumor invasion and metastasis . Evidences suggest that circulating anti-MUC1-IgG is a favorable prognostic factor for pancreatic cancer . Downregulation of MUC1 expression in human PC cell line S2-013 by RNAi significantly decreased proliferation in vitro and in nude mice . In a murine model, the use of MUC1-specific 90Yttrium-labelled moAb PAM4 in combination with gemcitabine as a radiosensitiser  increased inhibition of tumor growth and prolonged animal survival. To date, it is undergoing phase I trial for stage III or IV PC patients.
In vitro study showed that 213Bi-C595 was specifically cytotoxic to MUC1-expressing PC cells in a concentration-dependent manner compared to controls. 213Bi-C595 is a moAb targeting the protein core of MUC1, conjugated with the α-particle-emitting 213bismuth .
PankoMab (Glycotope, Germany) is a murine anti-human MUC-1 antibody that binds to a carbohydrate-induced conformational tumor epitope of MUC-1, greatly increasing its tumor specificity . PankoMab can induce ADCC of MUC-1 positive cells and can also induce death following internalization by inhibition of RNA polymerase when linked to β-amanitin. The humanized version of PankoMab has been shown to react to the tumor expressed MUC-1 in multiple human carcinomas, although no clinical trials have been published .
The epidermal growth factor receptor 2 (HER2), a transmembrane receptor tyrosine kinase, is overexpressed in up to 45% of pancreatic cancer. An anti-Her-2/neu antibody, known as Herceptin (Genentech Inc., CA, USA) or trastuzumab, has been used with some success to treat PC murine models. Treatments with trastuzumab prolonged survival and reduced liver metastasis in nude mice orthotopically challenged with human pancreatic tumor cell lines that expressed Her-2/neu at low levels. The pancreatic lines were sensitive to ADCC lysis by trastuzumab in vitro . Similar results were found when nude mice (challenged with Her-2/neu high expressing human PC cell lines) were treated with both trastuzumab and 5-fluorouracil . The combination of treatments significantly inhibited tumor growth compared with either treatment alone. When combined with matuzumab, an anti-EGFR antibody, trastuzumab treatment, resulted in inhibited PC growth in a nude mouse . Also, this combined treatment was more effective than treatment with either antibody alone or combined with gemcitabine .
Carcinoembryonic antigen (CEA), a member of a family of cell surface glycoproteins involved in cell adhesion, is frequently overexpressed in various types of human cancers. Many anti-CEA antibodies have been used for immunotherapy, such as hMN-14 (labetuzumab), which has been shown to induce ADCC in vitro with CEA+ colon tumor cells and inhibited growth of lung metastases in nude mice . A phase I/II trial with hMN-14 in PC patients has been completed but the results have not been published .
EGFR is a transmembrane glycoprotein receptor, overexpressed in 90% of pancreatic tumors , which induces tumor cell proliferation and neovascularization; also this expression is associated with worse prognosis [50, 51]. Blocking EGFR signaling decreases growth and metastasis of pancreatic tumor in animal models and enhances the effects of gemcitabine [52, 53].
Cetuximab (Erbitux or IMC-C225) is a chimeric monoclonal antibody generated from fusion of the variable region of the murine anti-EGFR monoclonal antibody M225 and the human IgG1 constant region. Promising laboratory results have led cetuximab to be tested in clinical trials. A phase III randomized study by Southwestern Oncology Group (SWOG) tested the efficacy of cetuximab and gemcitabine combination in patients with advanced PC. The median survival was 6 months in the gemcitabine arm and 6.5 months in the combination arm for an overall hazard ratio (HR) of 1.09 (). The corresponding progression free survival was 3 months and 3.5 months, respectively. The study failed to demonstrate a clinically significant advantage of the addition of cetuximab to gemcitabine . In an ongoing phase II trial with trimodal therapy of cetuximab, gemcitabine and intensity modulated radiotherapy (IMRT) for patients with advanced PC; there was no increase in toxicity profile . One-year survival was 57% while median survival has not been reached.
Matuzumab (EMD72000) is a humanized IgG1 monoclonal antibody to the human EGFR. Laboratory studies have shown promising inhibitory effects on tumor growth and angiogenesis, including L3.6pl in an orthotopic rat model . In a phase I study of combined treatment with matuzumab and gemcitabine, eight out of 12 patients with advanced pancreatic adenocarcinoma showed partial response or stable disease .
Vascular endothelial growth factor (VEGF) plays a pivotal role in the control of angiogenesis, tumor growth, and metastasis . VEGF and its receptors are overexpressed in PC and have been demonstrated to be a poor prognostic factor. There is suggestion that elevated serum VEGF levels correlate with tumor stage, disease recurrence, and survival . Development of therapeutic strategies directed towards the VEGF mediated signaling axis has been extensively tested in patients with advanced PC.
Bevacizumab (Avastin) is a recombinant humanized anti-VEGF monoclonal antibody. A pilot study demonstrated that bevacizumab, when added to gemcitabine in patients with metastatic PC, resulted in a significant improvement in response, survival, and progression-free survival . This was immediately followed by a phase III trial by CALGB comparing gemcitabine plus bevacizumab to gemcitabine plus placebo and showing no benefit for bevacizumab addition . The AviTa phase III trial that examined treatment with gemcitabine plus erlotinib with either bevacizumab or placebo has been closed. Bevacizumab, however, may have a role in palliative treatment of chemotherapy-resistant PC. In a case report, a patient with stage IV disease initially unresponsive to gemcitabine, 5-FU, irinotecan, and cisplatin subsequently responded with the addition of bevacizumab .
2.2. Cellular Mediated Immunity: Adoptive T Cell Transfer
Adoptive T cell transfer is a form of immunotherapy in which patient’s own T cells are expanded and reinfused into the patient. In particular, this method involves harvesting the patient’s peripheral blood T lymphocytes, stimulating and expanding the autologous tumor-reactive T cells using IL-2 and CD3-specific antibody, before subsequently transferring them back into the patient. Adoptive T cell therapy depends on the ability to optimally select or genetically engineer cells with targeted antigen specificity and then to induce the cell proliferation preserving their effector function and engraftment and homing abilities. Currently, there are no FDA-approved adoptive T cell therapy protocols for cancer, but T cell therapies have shown activity in mice models and in selected clinical applications. For example, adoptive transfer of telomerase-specific T cells was studied in a syngeneic PC murine model . T cells were produced in vitro by coculturing human lymphocytes with telomerase peptide-pulsed dendritic cells (DCs) or in vivo by injection of peptide with adjuvant into C57BL/6 mice. Telomerase is a reverse transcriptase that contains an RNA template used to synthesize telomeric repeats onto chromosomal ends. Activation of telomerase and its maintenance of telomeres play a role in immortalization of human cancer cells, as telomeres shrink after each cell division . Telomerase activity is found in 92–95% of pancreatic cancers [65, 66] and is associated with increased potential of invasion and metastasis and poor prognosis [67, 68]. Upregulation of telomerase may also be responsible for the development of chemotherapy resistance . Animals treated with these T cells showed significantly delayed disease progression .
Adoptive transfer of MUC1-specific cytotoxic T-lymphocytes (CTLs) was able to completely eradicate MUC1-expressing tumors in mice . In this perspective, in a clinical study, MUC-1-specific autologous T cells, isolated from patient PBMCs (peripheral blood mononuclear cells), were expanded by incubation with an MUC-1-presenting cell line prior to administration in PC patients. The mean survival time for unresectable patients in this study was 5 months . However, patients with resectable pancreatic cancer had 1-, 2-, and 3-year survival rates of 83.3, 32.4, and 19.4%, respectively, and a mean survival time of 17.8 months. In a similar study, Kondo et al. isolated adherent cells from patient PBMCs to generate mature DCs that were then pulsed with MUC-1 peptide. The pulsed DCs were administered, along with autologous expanded MUC-1-specific T cells, to patients with unresectable or recurrent pancreatic cancer. Remarkably, a complete response was observed in one patient with lung metastases, and the MS time of the whole group was 9.8 months, suggesting that the addition of pulsed DCs may have improved the outcome .
3. Active Immunotherapy: Vaccine Strategies
Vaccination involves administering a tumor antigen with the aim of stimulating tumor-specific immunity. Antigens could be delivered in the form of DNA or peptides, as well as tumor cells or antigen-pulsed DCs. To be considered an ideal tumor vaccine candidate, expression of the antigen must be restricted to the tumor or only minimally expressed elsewhere in the body. Table 1 summaries a list of major candidate pancreas tumor-associated antigens for immune targeting. Additional synergistic help is added to elicit a more vigorous and effective immune response, such as cytokines and immunostimulating compounds. Vaccination against tumor antigens is an attractive approach to adjuvant treatment aftersurgery, when tumor-induced immune suppression is minimal [73–75].
3.1. Vaccines Using Whole Cells
The simplest vaccine approach that has been applied to cancer is the inoculation of patients with irradiated tumor cells. This approach remains a potent vehicle for generating antitumor immunity because tumor cells express all relevant candidate TAAs, including both known and unidentified. In the clinical setting, the use of autologous tumor cell depends on the availability of an adequate number of them. As only 10–15% of PC patients diagnosed are eligible for surgical, autologous pancreatic cancer cells may not be provided in most of the patients. Moreover, even if the patients are treated by surgical resection, it is difficult to prepare sufficient numbers of tumor cells due to the length of culture time and risk of contamination [76, 77]. To elude this problem, allogeneic tumor cell lines may be used instead of autologous tumor cells . This strategy has many advantages: (1) specific TAAs do not need to be identified for vaccination, (2) allogeneic tumor cell lines are well characterized as TAAs source, (3) allogeneic tumor cell lines can grow well in vitro; thus, there is no limiting factor for preparation of tumor cells, (4) it is not necessary to determine HLA typing of patients and allogeneic tumor cells, because autologous DCs can process and present multiple TAAs from allogeneic tumor cells owing to cross-presentation in the context of appropriate MHC class I and II alleles [75, 79], (5) polyclonal antigen-specific T cells (CD4+/CD8+) can be generated, which may protect against tumor escape variants, and (6) the tumor cell vaccine platform can be easily modified. For example, tumor cells can be transduced to express immunomodulatory cytokines such as granulocyte macrophage colony-stimulating factor (GM-CSF), which has shown significant antitumor effect in vivo . GM-CSF is an important growth factor for granulocytes and monocytes and has a crucial role in the growth and differentiation of DCs. In a phase I clinical trial, Jaffee et al.  used allogeneic GM-CSF-secreting whole-cell tumor vaccine for pancreatic cancer, based on the concept that the GM-CSF localization in the implanted tumor environment together with the shared tumor antigen expressed by the primary cancer would effectively induce an antitumor immune response. In this study two PC cell lines (PANC 10.05 and PANC 6.03) were used as the vaccine, both genetically modified to express GM-CSF and then irradiated to prevent further cell division. 14 PC patients who had undergone pancreatic duodenectomy eight weeks before were given variable doses of the vaccine intradermally. Three of the eight patients who received ≥10 × 107 vaccine cells developed postvaccination delayed-type hypersensitivity (DTH) responses associated with increased disease-free survival time and remained disease free for longer than 25 months after diagnosis. Side effects were mainly limited to local skin reactions at the site of vaccination.
In a recently completed phase II study 60 patients with resected pancreatic adenocarcinoma received five treatments of 2.5 × 108 vaccine cells, together with 5-FU and radiotherapy . The reported MS was 26 months, with a one- and two-year survival of 88% and 76%, respectively. In these two studies, a PC cell vaccine induced a CD8+ T cell response, specific to mesothelin, regardless of HLA match between the tumor vaccine and recipient—demonstrating that cross-priming had occurred [80, 82]. Mesothelin is a particularly promising cancer vaccine target owing to its low level of expression in nontumor tissues and high levels of expression in pancreatic as well as other cancers (i.e., ovarian) . Laheru et al.  administrated GMCSF-secreting allogeneic PC cells in sequence with cyclophosphamide in patients with advanced pancreatic cancer. The approach showed minimal treatment-related toxicity and mesothelin-specific T cell responses. Moreover, combination of the vaccine and cyclophosphamide resulted in MS in a gemcitabine-resistant population similar to chemotherapy alone. It was also reported that combination of the vaccines and chemoradiation demonstrated an overall survival that compares favorably with published data for resected pancreas cancer .
Tumor cell vaccines have also been modified to express epitopes, which increase antibody-mediated uptake by DCs. Normally, MUC-1 expressed on tumors is immunogenic owing to overexpression and tumor-restricted hypoglycosylation . The NewLink Genetics Corporation (IA, USA) has developed a whole-cell vaccine expressing MUC-1 modified to express α-gal epitopes, which is the focus of multiple clinical trials [87–90]. This vaccine takes advantage of anti-α-gal antibodies that are found in most people due to exposure to gastrointestinal flora, resulting in increased uptake of the vaccine in an antibody-dependent manner . In murine models, the addition of such α-gal epitopes to a Muc-1+ PC whole-cell vaccine resulted in increased production of anti-Muc-1 antibodies, enhanced tumor-specific T cell responses, and increased survival after challenge with Muc-1+ B16 cells in α-gal knockout mice, previously sensitized to α-gal .
3.2. Peptide Vaccines
Peptide-based cancer vaccines are preparations made from antigenic protein fragments (called epitopes), that represent the minimal immunogenic region of antigens [93, 94], designed to enhance the T cell response, especially the CD8+. Induction of CTLs needs peptides derived from TAAs to be presented on the surface of APCs (antigens presenting cells), such as DCs, in the context of HLA molecules. The major advantages of peptide vaccines are that they are simple, stable, safe, economical, and do not require manipulation of patient tissues, whose availability may be limited. However, there are also several obstacles that limit the widespread usefulness of peptide vaccines: (1) a limited number of known synthesized short peptides cannot be available in many HLA molecules [95–97], (2) impaired function of APCs in patients with advanced pancreatic cancer [76, 98], (3) CTLs may be ineffective in reacting with PC cells downregulated by certain tumor antigens and MHC class I molecules, which may appear during the course of tumor progression , (4) regulatory T cells (Tregs) or MDSCs (myeloid-derived suppressor cells) in tumor environment produce immunosuppressive cytokines such as IL-10 and TGF-β .
Anyway, a number of peptide vaccines have undergone phase I/II clinical trials [12, 101], showing encouraging results, due to their ability to produce cancer-specific responses in PC patients (Table 2). In a phase I study, vaccination with a 100 mer peptide of the MUC-1 extracellular tandem repeat generated a MUC-1-specific T cell response in some PC patients with two of the 15 patients alive at 61 months . Moreover, in a separate phase I clinical trial using the same peptide vaccine, the production of anti-MUC-1 circulating antibodies was detected in patients with inoperable PC, although no significant impact on survival was discovered .
In a phase I trial, Miyazawa et al. administered a peptide vaccine for human VEGF receptor, (VEGFR)2-169 epitope, in patients with advanced PC, in combination with gemcitabine, observing an antigen-specific DTH and VEGFR2-specific CD8+ cells in 61% patients, with an overall MS time of 8.7 months . A randomized, placebo-controlled, multicenter, phase II/III study of this VEGFR2–169 peptide vaccine therapy, combined with gemcitabine, is currently underway in patients with unresectable advanced or recurrent PC . In similar studies, a telomerase-based vaccine, consisting of the human telomerase reverse transcriptase (GV1001) peptide, was found to induce a telomerase-specific immune response in 63% of evaluable patients, as measured by DTH in unresectable PC. Those with a positive DTH were found to live longer than those that did not have a positive DTH . In addition, augmented immune responses and prolonged survival were observed following vaccination of advanced PC patients with telomerase peptide and GM-CSF . More recently, a phase III clinical trial was performed in which the effect of gemcitabine treatment on survival was compared with gemcitabine treatment in combination with GV1001 therapy in unresectable and metastatic PC patients . However, the trial was terminated when no survival benefit was found.
The most interesting results have come from studies of K-Ras-targeted peptide vaccines. Gjertsen et al.  first reported mutant K-ras peptide vaccines for PC. In a phase I/II trial involving 48 PC patients, they studied ras peptide in combination with GM-CSF, since native epitopes have relatively low immunogenicity . Peptide-specific immunity was induced in 58% of patients. Of patients with advanced disease, those who responded to treatment showed increased survival compared to nonresponders. Recently, another group reported that vaccination of 24 PC patients with K-ras peptide in combination with GM-CSF proved to be safe without tumor regression . In another pilot vaccine study, pancreatic and colorectal patients were vaccinated with K-Ras peptides containing patient-specific mutations. Three of the five PC patients displayed an antigen-specific immune response to a K-Ras . Disease progression was observed in the two PC patients that did not respond to the vaccine, with the responders having no evidence of disease. Of the PC patients, a mean disease-free survival of 35.2 months and a mean overall survival of 44.4 months were observed. Such results with peptide vaccines are highly encouraging.
The more attractive peptide-based vaccines may be synthetic long peptides to induce antigen-specific polyclonal CD8+ and CD4+ T cells . Long synthetic peptides cannot bind directly on MHC class I or II molecules, but they need to be processed and presented by DCs. So, the long peptide vaccines can present MHC class I- and II-restricted epitopes for long time, thus eliciting both CD4+- and CD8+-mediated immune recognition  and probably inducing a robust therapeutic T cell response. In a phase I study using long synthetic mutant ras peptides, Wedén et al.  treated 23 patients who were vaccinated after surgical PC resection. Long-term immunological memory responses to the vaccines were observed. Strikingly, 10-year survival was 20% (four patients out of 20 evaluable) versus zero (0/87) in a cohort of nonvaccinated patient treated in the same period.
To increase the immunogenicity of peptide vaccines, some groups have mutated key anchor residues in the peptides such that binding to MHC-I molecules, and consequently the presentation to CD8+ T cells, is increased. This is particularly important when vaccinating against TAAs, as they are often weak or only intermediate binders to HLA molecules [112–116]. An MUC-1 peptide vaccine modified in this way was shown to enhance production of IFN-γ from patient and normal donor T cells. MUC-1-specific T cell clones, generated by stimulation with this peptide, could lyse targets pulsed with native Muc-1 epitope as well as HLA-A2+ MUC-1+ human tumor cells in vitro . Notably, one case has been reported in which vaccination with a modified HLA-A2-restricted survivin peptide resulted in remission of liver metastasis in one PC patient .
Another approach in cancer peptide-vaccination consists in using personalized peptide vaccines based on the tumor-antigen epitopes that are most immunogenic for a particular patient. In a phase I clinical trial, Yanagimoto et al. applied this strategy, in combination with gemcitabine therapy, to pancreatic cancer. Prior to vaccination, T cells from patient PBMCs were screened against a panel of tumor antigen-derived peptides. Patients were vaccinated only with the peptides to which they had a response . An increase in tumor antigen-specific T cell responses was observed from the 13 evaluable patients with no correlation to clinical responses or humoral responses following vaccination, although 11 patients experienced either reduction in tumor size. Median survival time was 7.6 months. A similar phase II study was published in 2010 by the same group, showing an MS time of 9 months and a 1-year survival of 38% .
3.3. DNA Vaccination
Vaccination with DNA represents a simple vehicle for in vivo transfection and antigen production. A DNA vaccine is composed of a plasmid DNA that encodes for a TAA under the control of a mammalian promoter and can be easily produced in the bacteria . It can be administered to humans via intramuscular injection with or without electroporation. Compared with cell-based vaccines, this vaccination strategy offers more advantages; in fact, while cell-based vaccines become less effective over time because the induced immune system recognizes them as foreign and quickly destroys them, DNA vaccines can provide prolonged antigen expression, leading to amplification of immune responses and inducing memory responses against weakly immunogenic TAAs. Moreover, as DNA might be taken up by cells and the encoded antigen is processed through both endogenous and exogenous pathways, DNA vaccines administered via intramuscular injection allow for an immune response to multiple potential epitopes within an antigen to be generated regardless of the recipient’s MHC profile . Actually DNA vaccines are ongoing trials in different tumors [121–123] and being studied in murine models of pancreatic cancer. In a murine PC study, an MUC-1 DNA vaccine was able to induce a significant MUC-1-specific CTL response and had both prophylactic and therapeutic effects in tumor-bearing mice . Similarly, in another PC murine model, vaccination with either murine or human full-length survivin DNA generated an antitumor-specific response, increased infiltration of tumor with lymphocytes and increased survival . Furthermore, Gaffney et al. studied the mesothelin DNA vaccine in combination with the antiglucocorticoid-induced TNF receptor antibody (anti-GITR) in mice with syngeneic mesothelin-expressing pancreatic cancer . 50% of animals treated with mesothelin were tumor free 25 days after tumor injection compared to 0% of nontreated mice. This increased to 94% with the addition of anti-GITR. The agonist anti-GITR served to enhance T cell-mediated response of the vaccine [127, 128].
3.4. Antigen-Pulsed DCs
Antigen-specific T cell responses are initiated by DCs. They capture antigens secreted or shed by tumor cells and present peptides in association with the MHC class I and II molecules. This results in the expression and upregulation of cytokines and costimulatory molecules which in turn stimulate CD4+ and CD8+ T cells to mount an antitumor response . Therefore, a major area of investigation in cancer immunotherapy involves the design of DCs-based cancer vaccines . Autologous DCs can be used in tumor vaccination (1) pulsed with synthetic peptide derived from the known tumor antigens , tumor cell lysates , or apoptotic tumor cells , (2) transfected with whole-tumor mRNA  or with mRNA or cDNA of a specific antigen  and (3) fused with tumor cells to induce antigen-specific polyclonal CTL responses .
DC-based vaccines have been used in different PC studies. Schmidt et al. intratumorally vaccinated with whole tumor mRNA transfected DCs and found an antitumor-specific immune response and significantly decreased tumor volume in a murine PC model . Apoptotic PC lysates have also been evaluated as a source of antigens and have been demonstrated to elicit stronger antitumor lytic activity when used to stimulate autologous human CD8+ T cells in vitro compared with those stimulated with tumor lysate-pulsed DCs . In cases in which an immunogenic tumor antigen is known, autologous DCs have been transfected with or virally transduced to express, the mRNA or cDNA of a specific tumor antigen (Table 3). A vaccine consisting of liposomal MUC1-transfected autologous DCs was evaluated in a clinical phase I/II trial. In MUC1 peptide-loaded DC vaccines in PC patients following resection of their primary tumors, four of the 12 patients followed for over four years were alive, all without evidence of recurrence . Moreover, MUC1-specific immune responses were also observed even in patients with pretreated and advanced disease, following immunization with DCs transfected with MUC1 cDNA . This technique does not require that the exact immunogenic epitopes of the antigen be identified, as full-length protein is transfected.
In another study, three patients with resected PC following neoadjuvant chemoradiotherapy were given monthly injections of autologous, monocyte-derived DCs loaded with the mRNA of CEA for six months . No toxicities were reported, and all patients remained disease free for more than 30 months from diagnosis.
Pulse can also be performed with peptides from multiple tumor antigens, as was performed in a Phase I clinical study by Carbone et al. Patients with various cancers, including pancreatic cancer, immunized with p53 and K-ras peptide-pulsed PBMCs, saw increased survival . In addition, autologous DCs, virally transduced to express IL-12, have also been used in cancer treatment. One PC patient receiving this treatment had a partial response in studies by Mazzolini et al. . As the treatment DCs were not loaded with tumor antigens, cross-presentation of tumor antigens must have occurred. Moreover, DCs have been fused with tumor cells to induce antigen-specific polyclonal CTL responses . In the DC/tumor cell fusion approach, whole TAAs including those known and those yet unidentified are processed endogenously and presented by MHC class I and II pathways in the context of costimulatory signals [144–146]. In particular, this technique has been used to treat mice in a PC model, resulting in the generation of CD8+ T cells with tumor-specific cytolytic activity and tumor rejection .
Pancreatic cancer is a dismal disease that has a high morbidity and mortality, and at present there are not effective chemotherapeutic treatments, especially for patients with advanced and metastatic diseases. For all these reasons Alphais of prime importance to investigate new pancreatic cancer treatments. In this paper we have analyzed the various strategies of the immunotherapeutic approach, some of which are still used in animal models; others are already being exploited in clinical trials. Immunotherapy is certainly a promising treatment for pancreatic cancer, because it is highly specific for cancer cells and therefore without the side effects associated with traditional chemotherapy. But at the moment there are not antigens expressed only by PC cells; in fact the antigens used as the target of immunotherapic treatments are self-protein or overexpressed [65, 148–150] in tumor cells or present in acetylated form , with the risk of autoimmune phenomena. However, the data obtained in different clinical trials showed an increase in the survival of patients treated with PC immunotherapy alone [72, 105, 108, 111] or in combination with chemotherapy treatments [12, 54, 101], with very minimal autoimmune manifestations. In conclusion we can say that immunotherapy may be included among the future treatments for pancreatic cancer, especially for inoperable patients, but for the effectiveness of this innovative treatment is essential to overcome some obstacles: (a) finding specific markers for pancreatic cancer cells, (b) mitigating the immune suppressive effects of tumor cells, (c) early diagnosis of the tumor so as to act in a timely manner before the cancer spreads in other locations.
The authors thank Italian Ministry of University and Research and Ente Cassa di Risparmio di Firenze for supporting their studies.
- M. W. Saif, “Pancreatic neoplasm in 2011: an update,” Journal of the Pancreas, vol. 12, no. 4, pp. 316–321, 2011.
- Surveillance Epidemiology and End Results (SEER), U.S. Cancer Statistics: 1999–2007 Incidence and Mortality Report, http://www.seer.cancer.gov/publications/uscs.html.
- M. W. Saif, “Controversies in the adjuvant treatment of pancreatic adenocarcinoma,” Journal of the Pancreas, vol. 8, no. 5, pp. 545–552, 2007.
- H. A. Burris III, M. J. Moore, J. Andersen et al., “Improvements in survival and clinical benefit with gemcitabine as first-line therapy for patients with advanced pancreas cancer: a randomized trial,” Journal of Clinical Oncology, vol. 15, no. 6, pp. 2403–2413, 1997.
- M. J. Moore, D. Goldstein, J. Hamm et al., “Erlotinib plus gemcitabine compared with gemcitabine alone in patients with advanced pancreatic cancer: a phase III trial of the National Cancer Institute of Canada Clinical Trials Group,” Journal of Clinical Oncology, vol. 25, no. 15, pp. 1960–1966, 2007.
- J. Yokokawa, C. Palena, P. Arlen et al., “Identification of novel human CTL epitopes and their agonist epitopes of mesothelin,” Clinical Cancer Research, vol. 11, no. 17, pp. 6342–6351, 2005.
- M. H. Andersen, L. O. Pedersen, J. C. Becket, and P. T. Straten, “Identification of a cytotoxic T lymphocyte response to the apoptosis inhibitor protein survivin in cancer patients,” Cancer Research, vol. 61, no. 3, pp. 869–872, 2001.
- F. M. Johnston, M. C. B. Tan, B. R. Tan Jr. et al., “Circulating mesothelin protein and cellular antimesothelin immunity in patients with pancreatic cancer,” Clinical Cancer Research, vol. 15, no. 21, pp. 6511–6518, 2009.
- Y. Kotera, J. D. Fontenot, G. Pecher, R. S. Metzgar, and O. J. Finn, “Humoral immunity against a tandem repeat epitope of human mucin MUC-1 in sera from breast, pancreatic, and colon cancer patients,” Cancer Research, vol. 54, no. 11, pp. 2856–2860, 1994.
- B. Kubuschok, F. Neumann, R. Breit et al., “Naturally occurring T-cell response against mutated p21 Ras oncoprotein in pancreatic cancer,” Clinical Cancer Research, vol. 12, no. 4, pp. 1365–1372, 2006.
- L. Wenandy, R. B. Sørensen, L. Sengeløv, I. M. Svane, P. T. Straten, and M. H. Andersen, “The immunogenicity of the hTERT540-548 peptide in cancer,” Clinical Cancer Research, vol. 14, no. 1, pp. 4–7, 2008.
- H. Yanagimoto, T. Mine, K. Yamamoto et al., “Immunological evaluation of personalized peptide vaccination with gemcitabine for pancreatic cancer,” Cancer Science, vol. 98, no. 4, pp. 605–611, 2007.
- Y. Oji, S. Nakamori, M. Fujikawa et al., “Overexpression of the Wilms' tumor gene WT1 in pancreatic ductal adenocarcinoma,” Cancer Science, vol. 95, no. 7, pp. 583–587, 2004.
- M. Ueda, Y. Miura, O. Kunihiro et al., “MUC1 overexpression is the most reliable marker of invasive carcinoma in intraductal papillary-mucinous tumor (IPMT),” Hepato-Gastroenterology, vol. 52, no. 62, pp. 398–403, 2005.
- K. Seki, T. Suda, Y. Aoyagi, et al., “Diagnosis of pancreatic adenocarcinoma by detection of human telomerase reverse transcriptasemessenger RNA in pancreatic juice with sample qualification,” Clinical Cancer Research, vol. 7, no. 7, pp. 1976–1981, 2001.
- M. K. Gjertsen, A. Bakka, J. Breivik et al., “Vaccination with mutant ras peptides and induction of T-cell responsiveness in pancreatic carcinoma patients carrying the corresponding RAS mutation,” The Lancet, vol. 346, no. 8987, pp. 1399–1400, 1995.
- M. Wobser, P. Keikavoussi, V. Kunzmann, M. Weininger, M. H. Andersen, and J. C. Becker, “Complete remission of liver metastasis of pancreatic cancer under vaccination with a HLA-A2 restricted peptide derived from the universal tumor antigen survivin,” Cancer Immunology, Immunotherapy, vol. 55, no. 10, pp. 1294–1298, 2006.
- K. Yamaguchi, M. Enjoji, and M. Tsuneyoshi, “Pancreatoduodenal carcinoma: a clinicopathologic study of 304 patients and immunohistochemical observation for CEA and CA19-9,” Journal of Surgical Oncology, vol. 47, no. 3, pp. 148–154, 1991.
- M. Komoto, B. Nakata, R. Amano et al., “HER2 overexpression correlates with survival after curative resection of pancreatic cancer,” Cancer Science, vol. 100, no. 7, pp. 1243–1247, 2009.
- H. Maacke, A. Kessler, W. Schmiegel et al., “Overexpression of p53 protein during pancreatitis,” British Journal of Cancer, vol. 75, no. 10, pp. 1501–1504, 1997.
- P. Cappello, B. Tomaino, R. Chiarle et al., “An integrated humoral and cellular response is elicited in pancreatic cancer by α-enolase, a novel pancreatic ductal adenocarcinoma-associated antigen,” International Journal of Cancer, vol. 125, no. 3, pp. 639–648, 2009.
- Y. Hamanakai, Y. Suehiro, M. Fukui, K. Shikichi, K. Imai, and Y. Hinoda, “Circulating anti-MUC1 IGG antibodies as a favorable prognostic factor for pancreatic cancer,” International Journal of Cancer, vol. 103, no. 1, pp. 97–100, 2003.
- F. Pagès, J. Galon, M. C. Dieu-Nosjean, E. Tartour, C. Sautès-Fridman, and W. H. Fridman, “Immune infiltration in human tumors: a prognostic factor that should not be ignored,” Oncogene, vol. 29, no. 8, pp. 1093–1102, 2010.
- X. R. Jiang, A. Song, S. Bergelson et al., “Advances in the assessment and control of the effector functions of therapeutic antibodies,” Nature Reviews Drug Discovery, vol. 10, no. 2, pp. 101–111, 2011.
- I. Hellstrom, E. Friedman, T. Verch et al., “Anti-mesothelin antibodies and circulating mesothelin relate to the clinical state in ovarian cancer patients,” Cancer Epidemiology Biomarkers and Prevention, vol. 17, no. 6, pp. 1520–1526, 2008.
- M. Ho, R. Hassan, J. Zhang et al., “Humoral immune response to mesothelin in mesothelioma and ovarian cancer patients,” Clinical Cancer Research, vol. 11, no. 10, pp. 3814–3820, 2005.
- R. Hassan, T. Bera, and I. Pastan, “Mesothelin: a new target for immunotherapy,” Clinical Cancer Research, vol. 10, no. 12 I, pp. 3937–3942, 2004.
- P. Argani, C. Iacobuzio-Donahue, B. Ryu et al., “Mesothelin is overexpressed in the vast majority of ductal adenocarcinomas of the pancreas: identification of a new pancreatic cancer marker by serial analysis of gene expression (SAGE),” Clinical Cancer Research, vol. 7, no. 12, pp. 3862–3868, 2001.
- R. Hassan, Z. G. Laszik, M. Lerner, M. Raffeld, R. Postier, and D. Brackett, “Mesothelin is overexpressed in pancreaticobiliary adenocarcinomas but not in normal pancreas and chronic pancreatitis,” American Journal of Clinical Pathology, vol. 124, no. 6, pp. 838–845, 2005.
- R. Hassan, S. Bullock, A. Premkumar et al., “Phase I study of SS1P, a recombinant anti-mesothelin immunotoxin given as a bolus I.V. infusion to patients with mesothelin-expressing mesothelioma, ovarian, and pancreatic cancers,” Clinical Cancer Research, vol. 13, no. 17, pp. 5144–5149, 2007.
- D. Filpula and H. Zhao, “Releasable PEGylation of proteins with customized linkers,” Advanced Drug Delivery Reviews, vol. 60, no. 1, pp. 29–49, 2008.
- R. J. Kreitman, R. Hassan, D. J. FitzGerald, and I. Pastan, “Phase I trial of continuous infusion anti-mesothelin recombinant immunotoxin SS1P,” Clinical Cancer Research, vol. 15, no. 16, pp. 5274–5279, 2009.
- D. K. Armstrong, D. Laheru, W. W. Ma, et al., “A phase 1 study of MORAb-009, a monoclonal antibody against mesothelin in pancreatic cancer, mesothelioma and ovarian adenocarcinoma,” Journal of Clinical Oncology, vol. 25, no. 18S, Article ID 14041, 2007.
- Y. Feng, D. Xiao, Z. Zhu et al., “A novel human monoclonal antibody that binds with high affinity to mesothelin-expressing cells and kills them by antibody-dependent cell-mediated cytotoxicity,” Molecular Cancer Therapeutics, vol. 8, no. 5, pp. 1113–1118, 2009.
- M. Ho, M. Feng, R. J. Fisher, C. Rader, and I. Pastan, “A novel high-affinity human monoclonal antibody to mesothelin,” International Journal of Cancer, vol. 128, no. 9, pp. 2020–2030, 2011.
- H. Yamasaki, S. Ikeda, M. Okajima et al., “Expression and localization of MUC1, MUC2, MUC5AC and small intestinal mucin antigen in pancreatic tumors,” International Journal of Oncology, vol. 24, no. 1, pp. 107–113, 2004.
- C. F. Qu, Y. Li, Y. J. Song et al., “MUC1 expression in primary and metastaticpancreatic cancer cells for in vitro treatment by 213Bi-C595 radioimmunoconjugate,” British Journal of Cancer, vol. 91, no. 12, pp. 2086–2093, 2004.
- E. Levi, D. S. Klimstra, A. Andea, O. Basturk, and N. V. Adsay, “MUC1 and MUC2 in pancreatic neoplasia,” Journal of Clinical Pathology, vol. 57, no. 5, pp. 456–462, 2004.
- H. Tsutsumida, B. J. Swanson, P. K. Singh et al., “RNA interference suppression of MUC1 reduces the growth rate and metastatic phenotype of human pancreatic cancer cells,” Clinical Cancer Research, vol. 12, no. 10, pp. 2976–2987, 2006.
- D. V. Gold, D. E. Modrak, K. Schutsky, and T. M. Cardillo, “Combined 90Yttrium-Dota-Labeled PAM4 antibody radioimmunotherapy and gemcitabine radiosensitization for the treatment of a human pancreatic cancer xenograft,” International Journal of Cancer, vol. 109, no. 4, pp. 618–626, 2004.
- A. Danielczyk, R. Stahn, D. Faulstich et al., “PankoMab: a potent new generation anti-tumour MUC1 antibody,” Cancer Immunology, Immunotherapy, vol. 55, no. 11, pp. 1337–1347, 2006.
- X. N. Fan, U. Karsten, S. Goletz, and Y. Cao, “Reactivity of a humanized antibody (hPankoMab) towards a tumor-related MUC1 epitope (TA-MUC1) with various human carcinomas,” Pathology Research and Practice, vol. 206, no. 8, pp. 585–589, 2010.
- G. Pratesi, G. Petrangolini, M. Tortoreto et al., “Antitumor efficacy of trastuzumab in nude mice orthotopically xenografted with human pancreatic tumor cells expressing low levels of HER-2/neu,” Journal of Immunotherapy, vol. 31, no. 6, pp. 537–544, 2008.
- H. Saeki, S. Yanoma, S. Takemiya et al., “Antitumor activity of a combination of trastuzumab (Herceptin) and oral fluoropyrimidine S-1 on human epidermal growth factor receptor 2-overexpressing pancreatic cancer,” Oncology Reports, vol. 18, no. 2, pp. 433–439, 2007.
- C. Larbouret, B. Robert, I. Navarro-Teulon et al., “In vivo therapeutic synergism of anti-epidermal growth factor receptor and anti-HER2 monoclonal antibodies against pancreatic carcinomas,” Clinical Cancer Research, vol. 13, no. 11, pp. 3356–3362, 2007.
- C. Larbouret, B. Robert, C. Bascoul-Mollevi et al., “Combined cetuximab and trastuzumab are superior to gemcitabine in the treatment of human pancreatic carcinoma xenografts,” Annals of Oncology, vol. 21, no. 1, pp. 98–103, 2010.
- R. D. Blumenthal, L. Osorio, M. K. Hayes, I. D. Horak, H. J. Hansen, and D. M. Goldenberg, “Carcinoembryonic antigen antibody inhibits lung metastasis and augments chemotherapy in a human colonic carcinoma xenograft,” Cancer Immunology, Immunotherapy, vol. 54, no. 4, pp. 315–327, 2005.
- A Phase I/II Study of Radioimmunotherapy with 90Y-Humanized MN-14 IgG Administered as a Single Dose to Patients with Refractory Advanced/Metastatic Pancreatic Carcinoma (NCT00041639), http://clinicaltrials.gov/.
- N. R. Lemoine, C. M. Hughes, C. M. Barton et al., “The epidermal growth factor receptor in human pancreatic cancer,” Journal of Pathology, vol. 166, no. 1, pp. 7–12, 1992.
- Y. Yamanaka, H. Friess, M. S. Kobrin, M. Buchler, H. G. Beger, and M. Korc, “Coexpression of epidermal growth factor receptor and ligands in human pancreatic cancer is associated with enhanced tumor aggressiveness,” Anticancer Research, vol. 13, no. 3, pp. 565–569, 1993.
- C. J. Bruns, C. C. Solorzano, M. T. Harbison et al., “Blockade of the epidermal growth factor receptor signaling by a novel tyrosine kinase inhibitor leads to apoptosis of endothelial cells and therapy of human pancreatic carcinoma,” Cancer Research, vol. 60, no. 11, pp. 2926–2935, 2000.
- S. S. W. Ng, M. S. Tsao, T. Nicklee, and D. W. Hedley, “Effects of the epidermal growth factor receptor inhibitor OSI-774, Tarceva, on downstream signaling pathways and apoptosis in human pancreatic adenocarcinoma,” Molecular Cancer Therapeutics, vol. 1, no. 10, pp. 777–783, 2002.
- J. Baselga and C. L. Arteaga, “Critical update and emerging trends in epidermal growth factor receptor targeting in cancer,” Journal of Clinical Oncology, vol. 23, no. 11, pp. 2445–2459, 2005.
- P. A. Philip, J. Benedetti, C. L. Corless et al., “Phase III study comparing gemcitabine plus cetuximab versus gemcitabine in patients with advanced pancreatic adenocarcinoma: Southwest oncology group-directed intergroup trial S0205,” Journal of Clinical Oncology, vol. 28, no. 22, pp. 3605–3610, 2010.
- R. Krempien, M. W. Munter, C. Timke, et al., “Cetuximab in combination with intensity modulated radiotherapy (IMRT) and gemcitabine for patients with locally advanced pancreatic cancer: a prospective phase II trial [PARC-Study ISRCTN56652283],” Journal of Clinical Oncology, vol. 25, no. 18S, article 4573, 2007.
- C. Bangard, A. Gossmann, A. Papyan, S. Tawadros, M. Hellmich, and C. J. Bruns, “Magnetic resonance imaging in an orthotopic rat model: blockade of epidermal growth factor receptor with EMD72000 inhibits human pancreatic carcinoma growth,” International Journal of Cancer, vol. 114, no. 1, pp. 131–138, 2005.
- U. Graeven, B. Kremer, T. Südhoff et al., “Phase I study of the humanised anti-EGFR monoclonal antibody matuzumab (EMD 72000) combined with gemcitabine in advanced pancreatic cancer,” British Journal of Cancer, vol. 94, no. 9, pp. 1293–1299, 2006.
- M. Korc, “Pathways for aberrant angiogenesis in pancreatic cancer,” Molecular Cancer, vol. 2, article 8, 2003.
- A. J. Karayiannakis, H. Bolanaki, K. N. Syrigos et al., “Serum vascular endothelial growth factor levels in pancreatic cancer patients correlate with advanced and metastatic disease and poor prognosis,” Cancer Letters, vol. 194, no. 1, pp. 119–124, 2003.
- H. L. Kindler, G. Friberg, D. A. Singh et al., “Phase II trial of bevacizumab plus gemcitabine in patients with advanced pancreatic cancer,” Journal of Clinical Oncology, vol. 23, no. 31, pp. 8033–8040, 2005.
- H. L. Kindler, D. Niedzwiecki, D. Hollis et al., “Gemcitabine plus bevacizumab compared with gemcitabine plus placebo in patients with advanced pancreatic cancer: phase III trial of the Cancer and Leukemia Group B (CALGB 80303),” Journal of Clinical Oncology, vol. 28, no. 22, pp. 3617–3622, 2010.
- H. W. Bruckner, V. R. Hrehorovich, and H. S. Sawhney, “Bevacizumab as treatment for chemotherapy-resistant pancreatic cancer,” Anticancer Research, vol. 25, no. 5, pp. 3637–3639, 2005.
- J. Schmidt, E. Ryschich, E. Sievers, I. G. H. Schmidt-Wolf, M. W. Büchler, and A. Märten, “Telomerase-specific T-cells kill pancreatic tumor cells in vitro and in vivo,” Cancer, vol. 106, no. 4, pp. 759–764, 2006.
- W. C. Hahn, “Role of telomeres and telomerase in the pathogenesis of human cancer,” Journal of Clinical Oncology, vol. 21, no. 10, pp. 2034–2043, 2003.
- E. Hiyama, T. Kodama, K. Shinbara et al., “Telomerase activity is detected in pancreatic cancer but not in benign tumors,” Cancer Research, vol. 57, no. 2, pp. 326–331, 1997.
- S. J. Myung, M. H. Kim, Y. S. Kim et al., “Telomerase activity in pure pancreatic juice for the diagnosis of pancreatic cancer may be complementary to K-ras mutation,” Gastrointestinal Endoscopy, vol. 51, no. 6, pp. 708–713, 2000.
- N. Sato, N. Maehara, K. Mizumoto et al., “Telomerase activity of cultured human pancreatic carcinoma cell lines correlates with their potential for migration and invasion,” Cancer, vol. 91, no. 3, pp. 496–504, 2001.
- S. J. Tang, J. A. Dumot, L. Wang et al., “Telomerase activity in pancreatic endocrine tumors,” American Journal of Gastroenterology, vol. 97, no. 4, pp. 1022–1030, 2002.
- N. Sato, K. Mizumoto, M. Kusumoto et al., “Up-regulation of telomerase activity in human pancreatic cancer cells after exposure to etoposide,” British Journal of Cancer, vol. 82, no. 11, pp. 1819–1826, 2000.
- P. Mukherjee, A. R. Ginardi, C. S. Madsen et al., “Mice with spontaneous pancreatic cancer naturally develop MUC-1-specific CTLs that eradicate tumors when adoptively transferred,” Journal of Immunology, vol. 165, no. 6, pp. 3451–3460, 2000.
- T. Kawaoka, M. Oka, M. Takashima et al., “Adoptive immunotherapy for pancreatic cancer: cytotoxic T lymphocytes stimulated by the MUC1-expressing human pancreatic cancer cell line YPK-1,” Oncology Reports, vol. 20, no. 1, pp. 155–163, 2008.
- H. Kondo, S. Hazama, T. Kawaoka et al., “Adoptive immunotherapy for pancreatic cancer using MUC1 peptide-pulsed dendritic cells and activated T lymphocytes,” Anticancer Research, vol. 28, no. 1B, pp. 379–387, 2008.
- T. Hensler, H. Hecker, K. Heeg et al., “Distinct mechanisms of immunosuppression as a consequence of major surgery,” Infection and Immunity, vol. 65, no. 6, pp. 2283–2291, 1997.
- G. Shakhar and S. Ben-Eliyahu, “Potential prophylactic measures against postoperative immunosuppression: could they reduce recurrence rates in oncological patients?” Annals of Surgical Oncology, vol. 10, no. 8, pp. 972–992, 2003.
- H. Weighardt, C. D. Heidecke, K. Emmanuilidis et al., “Sepsis after major visceral surgery is associated with sustained and interferon-γ-resistant defects of monocyte cytokine production,” Surgery, vol. 127, no. 3, pp. 309–315, 2000.
- S. Koido, E. Hara, S. Homma et al., “Dendritic/pancreatic carcinoma fusions for clinical use: comparative functional analysis of healthy-versus patient-derived fusions,” Clinical Immunology, vol. 135, no. 3, pp. 384–400, 2010.
- M. K. Gjertsen, T. Buanes, A. R. Rosseland et al., “Intradermal ras peptide vaccination with granulocyte-macrophage colony-stimulating factor as adjuvant: clinical and immunological responses in patients with pancreatic adenocarcinoma,” International Journal of Cancer, vol. 92, no. 3, pp. 441–450, 2001.
- S. Koido, E. Hara, S. Homma et al., “Dendritic cells fused with allogeneic colorectal cancer cell line present multiple colorectal cancer-specific antigens and induce antitumor immunity against autologous tumor cells,” Clinical Cancer Research, vol. 11, no. 21, pp. 7891–7900, 2005.
- H. Saito, P. Dubsky, C. Dantin, O. J. Finn, J. Banchereau, and A. K. Palucka, “Cross-priming of cyclin B1, MUC-1 and survivin-specific CD8+ T cells by dendritic cells loaded with killed allogeneic breast cancer cells,” Breast Cancer Research, vol. 8, no. 6, article R65, 2006.
- E. M. Jaffee, R. H. Hruban, B. Biedrzycki et al., “Novel allogeneic granulocyte-macrophage colony-stimulating factor-secreting tumor vaccine for pancreatic cancer: a phase I trial of safety and immune activation,” Journal of Clinical Oncology, vol. 19, no. 1, pp. 145–156, 2001.
- D. Laheru, C. Yeo, B. Biedrzycki, et al., “A safety and efficacy trial of lethally irradiated allogeneic pancreatic tumor cells transfected with the GM-CSF gene in combination with adjuvant chemoradiotherapy for the treatment of adenocarcinoma of the pancreas,” Journal of Clinical Oncology, vol. 25, no. 18S, article 3010, 2007.
- A. M. Thomas, L. M. Santarsiero, E. R. Lutz et al., “Mesothelin-specific CD8+ T cell responses provide evidence of in vivo cross-priming by antigen-presenting cells in vaccinated pancreatic cancer patients,” Journal of Experimental Medicine, vol. 200, no. 3, pp. 297–306, 2004.
- R. Hassan and M. Ho, “Mesothelin targeted cancer immunotherapy,” European Journal of Cancer, vol. 44, no. 1, pp. 46–53, 2008.
- D. Laheru, E. Lutz, J. Burke et al., “Allogeneic granulocyte macrophage colony-stimulating factor-secreting tumor immunotherapy alone or in sequence with cyclophosphamide for metastatic pancreatic cancer: a pilot study of safety, feasibility, and immune activation,” Clinical Cancer Research, vol. 14, no. 5, pp. 1455–1463, 2008.
- K. M. Hege, K. Jooss, and D. Pardoll, “GM-CSF gene-modifed cancer cell immunotherapies: of mice and men,” International Reviews of Immunology, vol. 25, no. 5-6, pp. 321–352, 2006.
- R. E. Beatson, J. Taylor-Papadimitriou, and J. M. Burchell, “MUC1 immunotherapy,” Immunotherapy, vol. 2, no. 3, pp. 305–327, 2010.
- A Phase I/II Study of an Antitumor Vaccination Using Alpha(1, 3) Galactosyltransferase Expressing Allogeneic Tumor Cells in Patients with Pancreatic Cancer (NCT00255827), http://clinicaltrials.gov/.
- A Phase II Study of Low Dose HyperAcute(R)-Pancreatic Cancer Vaccine in Subjects with Surgically Resected Pancreatic Cancer (NCT00614601), http://clinicaltrials.gov/.
- A Phase II Study of HyperAcute(R)-Pancreatic Cancer Vaccine in Subjects with Surgically Resected Pancreatic Cancer (NCT00569387), http://clinicaltrials.gov/.
- R. B. Mandell, R. Flick, W. R. Staplin et al., “The αgal hyperAcute technology: enhancing immunogenicity of antiviral vaccines by exploiting the natural αgal-mediated zoonotic blockade,” Zoonoses and Public Health, vol. 56, no. 6-7, pp. 391–406, 2009.
- B. A. Macher and U. Galili, “The Galα1,3Galβ1,4GlcNAc-R (α-Gal) epitope: a carbohydrate of unique evolution and clinical relevance,” Biochimica et Biophysica Acta, vol. 1780, no. 2, pp. 75–88, 2008.
- T. Deguchi, M. Tanemura, E. Miyoshi et al., “Increased immunogenicity of tumor-associated antigen, mucin 1, engineered to express α-Gal epitopes: a novel approach to immunotherapy in pancreatic cancer,” Cancer Research, vol. 70, no. 13, pp. 5259–5269, 2010.
- A. W. Purcell, J. McCluskey, and J. Rossjohn, “More than one reason to rethink the use of peptides in vaccine design,” Nature Reviews Drug Discovery, vol. 6, no. 5, pp. 404–414, 2007.
- M. S. Bijker, C. J. M. Melief, R. Offringa, and S. H. van der Burg, “Design and development of synthetic peptide vaccines: past, present and future,” Expert Review of Vaccines, vol. 6, no. 4, pp. 591–603, 2007.
- S. Kanodia and W. M. Kast, “Peptide-based vaccines for cancer: realizing their potential,” Expert Review of Vaccines, vol. 7, no. 10, pp. 1533–1545, 2008.
- C. J. Voskens, S. E. Strome, and D. A. Sewell, “Synthetic peptide-based cancer vaccines: lessons learned and hurdles to overcome,” Current Molecular Medicine, vol. 9, no. 6, pp. 683–693, 2009.
- S. Mocellin, P. Pilati, and D. Nitti, “Peptide-based anticancer vaccines: recent advances and future perspectives,” Current Medicinal Chemistry, vol. 16, no. 36, pp. 4779–4796, 2009.
- H. Yanagimoto, S. Takai, S. Satoi et al., “Impaired function of circulating dendritic cells in patients with pancreatic cancer,” Clinical Immunology, vol. 114, no. 1, pp. 52–60, 2005.
- T. A. Waldmann, “Immunotherapy: past, present and future,” Nature Medicine, vol. 9, no. 3, pp. 269–277, 2003.
- O. J. Finn, “Molecular origins of cancer: cancer immunology,” The New England Journal of Medicine, vol. 358, no. 25, pp. 2704–2715, 2008.
- M. Miyazawa, R. Ohsawa, T. Tsunoda et al., “Phase I clinical trial using peptide vaccine for human vascular endothelial growth factor receptor 2 in combination with gemcitabine for patients with advanced pancreatic cancer,” Cancer Science, vol. 101, no. 2, pp. 433–439, 2010.
- R. K. Ramanathan, K. M. Lee, J. McKolanis et al., “Phase I study of a MUC1 vaccine composed of different doses of MUC1 peptide with SB-AS2 adjuvant in resected and locally advanced pancreatic cancer,” Cancer Immunology, Immunotherapy, vol. 54, no. 3, pp. 254–264, 2005.
- K. Yamamoto, T. Ueno, T. Kawaoka et al., “MUC1 peptide vaccination in patients with advanced pancreas or biliary tract cancer,” Anticancer Research, vol. 25, no. 5, pp. 3575–3579, 2005.
- International Clinical Trials Registry Platform (ICTRP), http://apps.who.int/trialsearch/trial.aspx?trialid=JPRN-UMIN000001664.
- S. L. Bernhardt, M. K. Gjertsen, S. Trachsel et al., “Telomerase peptide vaccination of patients with non-resectable pancreatic cancer: a dose escalating phase I/II study,” British Journal of Cancer, vol. 95, no. 11, pp. 1474–1482, 2006.
- T. Buanes, J. Maurel, W. Liauw, M. Hebbar, and J. Nemunaitis, “A randomized Phase III study of gemcitabine (G) versus GV1001 in sequential combination with G in patients with unresectable and metastatic pancreatic cancer (PC),” Journal of Clinical Oncology, vol. 27, no. 15S, article 4601, 2009.
- G. K. Abou-Alfa, P. B. Chapman, J. Feilchenfeldt et al., “Targeting mutated K-ras in pancreatic adenocarcinoma using an adjuvant vaccine,” American Journal of Clinical Oncology, vol. 34, no. 3, pp. 321–325, 2011.
- A. Toubaji, M. Achtar, M. Provenzano et al., “Pilot study of mutant ras peptide-based vaccine as an adjuvant treatment in pancreatic and colorectal cancers,” Cancer Immunology, Immunotherapy, vol. 57, no. 9, pp. 1413–1420, 2008.
- C. J. M. Melief and S. H. van der Burg, “Immunotherapy of established (pre)malignant disease by synthetic long peptide vaccines,” Nature Reviews Cancer, vol. 8, no. 5, pp. 351–360, 2008.
- M. S. Bijker, S. J. F. van den Eeden, K. L. Franken, C. J. M. Melief, S. H. van der Burg, and R. Offringa, “Superior induction of anti-tumor CTL immunity by extended peptide vaccines involves prolonged, DC-focused antigen presentation,” European Journal of Immunology, vol. 38, no. 4, pp. 1033–1042, 2008.
- S. Wedén, M. Klemp, I. P. Gladhaug et al., “Long-term follow-up of patients with resected pancreatic cancer following vaccination against mutant K-ras,” International Journal of Cancer, vol. 128, no. 5, pp. 1120–1128, 2011.
- J. Begley and A. Ribas, “Targeted therapies to improve tumor immunotherapy,” Clinical Cancer Research, vol. 14, no. 14, pp. 4385–4391, 2008.
- L. Chapatte, M. Ayyoub, S. Morel et al., “Processing of tumor-associated antigen by the proteasomes of dendritic cells controls in vivo T-cell responses,” Cancer Research, vol. 66, no. 10, pp. 5461–5468, 2006.
- A. N. Houghton and J. A. Guevara-Patiño, “Immune recognition of self in immunity against cancer,” Journal of Clinical Investigation, vol. 114, no. 4, pp. 468–471, 2004.
- L. M. Weiner, R. Surana, and J. Murray, “Vaccine prevention of cancer: can endogenous antigens be targeted?” Cancer Prevention Research, vol. 3, no. 4, pp. 410–415, 2010.
- Z. Yu, M. R. Theoret, C. E. Touloukian et al., “Poor immunogenicity of a self/tumor antigen derives from peptide-MHC-I instability and is independent of tolerance,” Journal of Clinical Investigation, vol. 114, no. 4, pp. 551–559, 2004.
- K. Y. Tsang, C. Palena, J. Gulley, P. Arlen, and J. Schlom, “A human cytotoxic T-lymphocyte epitope and its agonist epitope from the nonvariable number of tandem repeat sequence of MUC-1,” Clinical Cancer Research, vol. 10, no. 6, pp. 2139–2149, 2004.
- H. Yanagimoto, H. Shiomi, S. Satoi et al., “A phase II study of personalized peptide vaccination combined with gemcitabine for non-resectable pancreatic cancer patients,” Oncology Reports, vol. 24, no. 3, pp. 795–801, 2010.
- R. G. Webster and H. L. Robinson, “DNA vaccines: a review of developments,” BioDrugs, vol. 8, no. 4, pp. 273–292, 1997.
- G. Eschenburg, A. Stermann, R. Preissner, H. A. Meyer, and H. N. Lode, “DNA vaccination: using the patient's immune system to overcome cancer,” Clinical and Developmental Immunology, vol. 2010, Article ID 169484, 14 pages, 2010.
- A Phase I Clinical Trial to Evaluate the Safety and Immunogenicity of a Mammaglobin-A DNA Vaccine in Breast Cancer Patients with Metastatic Disease (NCT00807781), http://clinicaltrials.gov/.
- DNA Vaccine Coding for the Rhesus Prostate Specific Antigen (rhPSA) and Electroporation in Patients with Relapsed Prostate Cancer, A Phase I/II Study (NCT00859729), http://clinicaltrials.gov/.
- A Phase Ia/Ib Study of the Safety and Immunogenicity of a Xenogeneic Tyrosinase DNA Vaccine Melanoma (NCT00471133), http://clinicaltrials.gov/.
- Y. Rong, D. Jin, W. Wu et al., “Induction of protective and therapeutic anti-pancreatic cancer immunity using a reconstructed MUC1 DNA vaccine,” BMC Cancer, vol. 9, article 191, 2009.
- K. Zhu, H. Qin, S. C. Cha et al., “Survivin DNA vaccine generated specific antitumor effects in pancreatic carcinoma and lymphoma mouse models,” Vaccine, vol. 25, no. 46, pp. 7955–7961, 2007.
- M. C. Gaffney, P. Goedegebuure, H. Kashiwagi et al., “DNA vaccination targeting mesothelin combined with anti-GITR antibody induces rejection of pancreatic adenocarcinoma,” American Association For Cancer Research, vol. 47, article 329a, 2006.
- A. D. Cohen, A. Diab, M. A. Perales et al., “Agonist anti-GITR antibody enhances vaccine-induced CD8+ T-cell responses and tumor immunity,” Cancer Research, vol. 66, no. 9, pp. 4904–4912, 2006.
- E. M. Esparza and R. H. Arch, “Glucocorticoid-induced TNF receptor functions as a costimulatory receptor that promotes survival in early phases of T cell activation,” Journal of Immunology, vol. 174, no. 12, pp. 7869–7874, 2005.
- R. M. Steinman, “The dendritic cell system and its role in immunogenicity,” Annual Review of Immunology, vol. 9, pp. 271–296, 1991.
- J. Banchereau and A. K. Palucka, “Dendritic cells as therapeutic vaccines against cancer,” Nature Reviews Immunology, vol. 5, no. 4, pp. 296–306, 2005.
- F. O. Nestle, S. Alijagic, M. Gilliet et al., “Vaccination of melanoma patients with peptide- or tumor lysate-pulsed dendritic cells,” Nature Medicine, vol. 4, no. 3, pp. 328–332, 1998.
- A. Mackensen, B. Herbst, J. L. Chen et al., “Phase I study in melanoma patients of a vaccine with peptide-pulsed dendritic cells generated in vitro from CD34+ hematopoietic progenitor cells,” International Journal of Cancer, vol. 89, no. 2, pp. 385–392, 2000.
- A. K. Palucka, H. Ueno, J. Connolly et al., “Dendritic cells loaded with killed allogeneic melanoma cells can induce objective clinical responses and MART-1 specific CD8+ T-cell immunity,” Journal of Immunotherapy, vol. 29, no. 5, pp. 545–557, 2006.
- E. Gilboa and J. Vieweg, “Cancer immunotherapy with mRNA-transfected dendritic cells,” Immunological Reviews, vol. 199, pp. 251–263, 2004.
- S. K. Nair, D. Boczkowski, M. Morse, R. I. Cumming, H. K. Lyerly, and E. Gilboa, “Induction of primary carcinoembryonic antigen (CEA)-specific cytotoxic T lymphocytes in vitro using human dendritic cells transfected with RNA,” Nature Biotechnology, vol. 16, no. 4, pp. 364–369, 1998.
- J. Gong, D. Chen, M. Kashiwaba, and D. Kufe, “Induction of antitumor activity by immunization with fusions of dendritic and carcinoma cells,” Nature Medicine, vol. 3, no. 5, pp. 558–561, 1997.
- T. Schmidt, C. Ziske, A. Märten et al., “Intratumoral immunization with tumor RNA-pulsed dendritic cells confers antitumor immunity in a C57BL/6 pancreatic murine tumor model,” Cancer Research, vol. 63, no. 24, pp. 8962–8967, 2003.
- M. Schnurr, C. Scholz, S. Rothenfusser et al., “Apoptotic pancreatic tumor cells are superior to cell lysates in promoting cross-priming of cytotoxic T cells and activate NK and γδT cells,” Cancer Research, vol. 62, no. 8, pp. 2347–2352, 2002.
- A. J. Lepisto, A. J. Moser, H. Zeh, et al., “A phase I/II study of a MUC1 peptide pulsed autologous dendritic cell vaccine as adjuvant therapy in patients with resected pancreatic and biliary tumors,” Cancer Therapy, vol. 6, no. B, pp. 955–964, 2008.
- G. Pecher, A. Häring, L. Kaiser, and E. Thiel, “Mucin gene (MUC1) transfected dendritic cells as vaccine: results of a phase I/II clinical trial,” Cancer Immunology, Immunotherapy, vol. 51, no. 11-12, pp. 669–673, 2002.
- M. A. Morse, S. K. Nair, D. Boczkowski et al., “The feasibility and safety of immunotherapy with dendritic cells loaded with CEA mRNA following neoadjuvant chemoradiotherapy and resection of pancreatic cancer,” International Journal of Gastrointestinal Cancer, vol. 32, no. 1, pp. 1–6, 2002.
- D. P. Carbone, I. F. Ciernik, M. J. Kelley et al., “Immunization with mutant p53- and K-ras-derived peptides in cancer patients: immune response and clinical outcome,” Journal of Clinical Oncology, vol. 23, no. 22, pp. 5099–5107, 2005.
- G. Mazzolini, C. Alfaro, B. Sangro et al., “Intratumoral injection of dendritic cells engineered to secrete interleukin-12 by recombinant adenovirus in patients with metastatic gastrointestinal carcinomas,” Journal of Clinical Oncology, vol. 23, no. 5, pp. 999–1010, 2005.
- S. Koido, E. Hara, S. Homma, K. Fujise, J. Gong, and H. Tajiri, “Dendritic/tumor fusion cell-based vaccination against cancer,” Archivum Immunologiae et Therapiae Experimentalis, vol. 55, no. 5, pp. 281–287, 2007.
- J. Gong, S. Koido, and S. K. Calderwood, “Cell fusion: from hybridoma to dendritic cell-based vaccine,” Expert Review of Vaccines, vol. 7, no. 7, pp. 1055–1068, 2008.
- S. Koido, E. Hara, S. Homma, T. Ohkusa, J. Gong, and H. Tajiri, “Cancer immunotherapy by fusions of dendritic cells and tumor cells,” Immunotherapy, vol. 1, no. 1, pp. 49–62, 2009.
- M. Yamamoto, T. Kamigaki, K. Yamashita et al., “Enhancement of anti-tumor immunity by high levels of Th1 and Th17 with a combination of dendritic cell fusion hybrids and regulatory T cell depletion in pancreatic cancer,” Oncology Reports, vol. 22, no. 2, pp. 337–343, 2009.
- F. V. Ona, N. Zamcheck, P. Dhar, T. Moore, and H. Z. Kupchik, “Carcinoembryonic antigen (CEA) in the diagnosis of pancreatic cancer,” Cancer, vol. 31, no. 2, pp. 324–327, 1973.
- S. Lei, H. E. Appert, B. Nakata, D. R. Domenico, K. Kim, and J. M. Howard, “Overexpression of HER2/neu oncogene in pancreatic cancer correlates with shortened survival,” International Journal of Pancreatology, vol. 17, no. 1, pp. 15–21, 1995.
- B. M. Ryan, N. O'Donovan, and M. J. Duffy, “Survivin: a new target for anti-cancer therapy,” Cancer Treatment Reviews, vol. 35, no. 7, pp. 553–562, 2009.
- W. Zhou, M. Capello, C. Fredolini et al., “Mass spectrometry analysis of the post-translational modifications of r-enolase from pancreatic ductal adenocarcinoma cells,” Journal of Proteome Research, vol. 9, no. 6, pp. 2929–2936, 2010.
- H. H. Emina and H. L. Kaufman, “CEA-based vaccines,” Expert Review of Vaccines, vol. 1, no. 1, pp. 49–63, 2002.
- C. K. Tang, M. Katsara, and V. Apostolopoulos, “Strategies used for MUC1 immunotherapy: human clinical studies,” Expert Review of Vaccines, vol. 7, no. 7, pp. 963–975, 2008.
- A. Scarpa, P. Capelli, K. Mukai et al., “Pancreatic adenocarcinomas frequently show p53 gene mutations,” The American Journal of Pathology, vol. 142, no. 5, pp. 1534–1543, 1993.
- F. Chen, W. Wang, and W. S. El-Deiry, “Current strategies to target p53 in cancer,” Biochemical Pharmacology, vol. 80, no. 5, pp. 724–730, 2010.
- C. Almoguera, D. Shibata, K. Forrester, J. Martin, N. Arnheim, and M. Perucho, “Most human carcinomas of the exocrine pancreas contain mutant c-K-ras genes,” Cell, vol. 53, no. 4, pp. 549–554, 1988.
- J. Itakura, T. Ishiwata, H. Friess et al., “Enhanced expression of vascular endothelial growth factor in human pancreatic cancer correlates with local disease progression,” Clinical Cancer Research, vol. 3, no. 8, pp. 1309–1316, 1997.
- M. Li, U. Bharadwaj, R. Zhang et al., “Mesothelin is a malignant factor and therapeutic vaccine target for pancreatic cancer,” Molecular Cancer Therapeutics, vol. 7, no. 2, pp. 286–296, 2008.
- H. J. Kang, S. K. Jung, S. J. Kim, and S. J. Chung, “Structure of human α-enolase (hENO1), a multifunctional glycolytic enzyme,” Acta Crystallographica D, vol. 64, no. 6, pp. 651–657, 2008.