Vaccine Therapy for Pancreatic Cancer: A Battle against Deadly Cancer

Pancreatic cancer is one of the deadliest human malignancies and little progress has been achieved in its treatment over the past decades. Historically, chemotherapy or radiotherapy did not provide significant survival benefit in advanced pancreatic cancer. Thus, new therapeutic approaches are needed. As there is strong evidence that vaccine therapy against pancreatic cancer elicits antitumor immune responses, scientists have tried to stimulate the antitumor activities of the immune system to fight against pancreatic cancer, but has not reached to an expected result. Pancreatic cancer activates both antitumor immune responses and immunosuppressive mechanisms leading to tumor development and progression. This action is achieved through mobilization and activation of immune suppressive cells (CAFs), tolerogenic DCs, MDSCs, TAMs, Treg cells and cancer cells-derived soluble factors that promote the induction of tolerance through the generation of CD4+αchain of IL-2R (CD25)+forkhead box P3 (Foxp3) subset. In addition, pancreatic cancer cells modulate the immune system and avoid detection by effector immune by production of immune suppressive cytokines (e.g., TGF-β, IL-10, and IL-6), by expressing surface molecules that mediate immune suppression (e.g., vascular endothelial growth factors (VEGFs), Fas ligand (Fas-L), programmed death-1 ligand (PD-L1), indolamine-2, and 3-dioxygenase (IDO). Identification of pancreatic cancer-associated antigens has spurred the development of vaccination-based strategies for treatment. Vaccine therapy relies on the administration of biological preparations that include an antigen that is specifically expressed by malignant cells, boosting the natural ability of the immune system to react against neoplastic cells. Potent vaccines stimulate antigen presentation by dendritic cells, hence driving the expansion of antigen-specific effector and memory T cells. Further, immune modulation and immunosuppressive environment by pancreatic cancer can be overcome by enhancing vaccine efficacy by combinatorial therapy. In this paper, we analyze recent preclinical and clinical efforts towards vaccine therapy for pancreatic cancer designed to target pancreatic cancer-associated antigens and to elicit an antitumor response in vivo. Journal of Cancer Science & Therapy J o u r na l o f C anc er ence & Tera p y


Introduction
Pancreatic cancer (PC) is the fourth leading cause of cancer related death in the United States and seventh in the China, with 45,220 new cases in 2013 and estimated 38,460 deaths in the USA [1,2]. Treatment of PC has become multimodal with chemotherapy, radiation and surgical resection in the hope of long term survival. The only chance for cure and long -term survival is microscopic negative margins with R0 surgical resection [3]. However, only 10% to 20% of patients with PC have resectable disease at the time of diagnosis, approximately 30 to 40 percent have locally advanced tumor and another 40 percent will have metastatic disease at the time of diagnosis [1,4], and thus palliative chemotherapy remains the only option for most of these patients [5]. The overall five-year survival probability is less than 5% for all stages combined [1,3,4,6]. Even with advances in surgical techniques most of the patients undergoing a complete surgical resection experience a recurrence [7]. Several autopsy studies suggest that 8%-15% of PC patients die with locally advanced disease and without metastatic spread [8].
There are only a few chemotherapeutic agents that have shown to be effective against PC to date, such as FOLFIRINOX regiment and more recently combination of gemcetabine and erlotinib has shown somehow better result in the treatment of PC [9,10]. The survival benefits for patients treated with these regimens are marginal and hence we are in urgent need of novel therapeutic approaches against PC.
As in recent years, researchers have grown deeper understanding in the field of cancer immunology and their antigens that have given great hope for new alternative treatments for a variety of solid tumors including PC. Emerging evidence supports a critical role for the immune system in PC tumor development, progression and eradication [11,12]. There is strong recent evidence that classical anticancer treatments heavily rely on the immune system for their effectiveness [13][14][15].

Dysfunctional Immune System in the PC-Tumor Development and Progression
It has been found that dysfunctional immune system in PC has a critical role in the development and progression of tumor [11,12]. It is known that both the innate and the adaptive immune system are active against human cancers [31]. However, cancer cells escape the innate and adaptive immune responses (immunosurveillance) by immunoselection (selection of nonimmunogenic tumor cell variants, also known as immunoediting) or immunosubversion (active suppression of the immune response) [32]. Anticancer function of the immune system is achieved by cytotoxic CD8 T cells, T helper-1 (Th1) cells, mature dendritic cells (DCs), activated pro-inflammatory macrophages (M1) and NK cells [33]. Under the tumor induced immunosuppressive environment T helper cells acquire a T helper cell type 2 phenotype (Th2), which does not support cytotoxic CD8 T cell responses and is resistance to tumors, macrophages (M1) switch to the immunosuppressive M2 state [34]. In addition, the environment in pancreatic cancer consist of not only cancer cells, but also immune suppressive cells such as cancer-associated fibroblasts (CAFs), tolerogenic DCs, myeloid-derived suppressor cells (MDSCs), immunosuppressive tumor-associated macrophages (TAMs), and T regulatory cells (Treg cells) which inhibit effector immune responses [35]. There are increasing evidences that cancer cells-derived soluble factors promote the induction of tolerance through the generation of CD4+αchain of IL-2R (CD25)+ forkhead box P3 (Foxp3)+ Treg subset, which is linked to compromised antitumor immune responses [36]. Pancreatic cancer cells modulate the immune system and avoid detection by effector immune by production of immune suppressive cytokines (e.g., TGF-β, IL-10, and IL-6), by expressing surface molecules that mediate immune suppression (e.g., vascular endothelial growth factors (VEGF s ), Fas ligand (Fas-L), programmed death-1 ligand (PD-L1), indolamine-2, and 3-dioxygenase (IDO) [35], and interference with MHC class I peptide presentation by down-regulation of MHC class I expression or disabling of the antigen degradation or antigen insertion into the MHC class I [34]. Thus, leading to tumor progression.

Cancer Vaccines Strategies
What determines whether cancer vaccines can become a success in human immunotherapy? Exactly, the same as required for infectious diseases, cancer has to be immunogenic and activate cytotoxic T cell responses. Consequently, cancer cells have to possess immunogenic antigens susceptible of being targeted by vaccination. Cancer vaccines are biological preparations that involve administering a tumor antigen with the aim of stimulating tumor-specific immunity. Antigen can be delivered by a number of ways in the form of whole tumor cell recombinant vaccines, dendritic cell (DC) vaccines that combine antigen with DCs to present to white cells, DNA vaccine by inserting viral, bacterial or yeast DNA into human or animal cells, or T-cell receptor peptide vaccines by inserting peptides to modulate cell-mediated immunity. 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. Vaccination against tumor antigens is an attractive approach to adjuvant treatment after surgery, when tumor-induced immune suppression is minimal [37]. Cancer vaccines were first approved for hepatocellular carcinoma and cervical cancer prevention. More recently, the first vaccine (Sipuleucel-T, Provenge) was approved for the treatment of hormone refractory prostate cancer [38].
Compared to all other standard modalities (surgery, chemotherapy, radiotherapy, and adaptive immunotherapy), an effective vaccine-based immune response against tumor may be the only cancer treatment with the potential benefit to last a lifetime. Theoretically, vaccinated patients could mount an immune response able to either cure tumor or keep it under constant restraint (i.e., immune surveillance), delaying tumor recurrence and prolonging survival.
For the development of efficient vaccine researchers have been using different strategies such as: -Cancer vaccine should seek for Tumor specific antigens and distinct from self-proteins.
-Selection of the appropriate adjuvant, molecules that activate antigen-presenting cells to stimulate an antigen specific cytotoxic T lymphocyte (CTL) mediated immune responses [39].
-Effective vaccine should seek to provide long term memory to prevent tumor recurrence which can activate both innate and adaptive immune systems [40].
-Efforts towards improving the clinical efficacy of immune therapy should involve strategies to neutralize or overcome immune suppression.

Vaccines for Pancreatic Cancer
Several vaccine therapy strategies are being actively tested in clinical trials. An overview of clinical trials in provided in Table 1.

A. Peptide vaccines
Peptide-based cancer vaccines are preparations made from antigenic protein fragments (called epitopes), that represent the minimal immunogenic region of antigens [41,42], 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.

KRAS vaccines:
The association of mutant Kras with pancreatic cancer was established two decades ago [43,44]. The most common activating point mutation involves the KRAS2 oncogene, on chromosome 12p, in over 90% of PC [45]. This is the highest fraction of K-ras alteration found in any human tumor type. Recent tumor genome sequencing studies has established the prevalence of mutant Kras in Pancreatic Intraepithelial Neoplasia (PanINs), the most common

GM-CSF secreting allogeneic PC cells and cyclophosphamide
Ongoing precursor lesions and in pancreatic cancer with increased precision [46][47][48]. In a Phase I/II study, the administration of synthetic KRASderived peptides in unresectable pancreatic cancer patients resulted in an immune response in 2 out of 5 individuals [49]. Since native epitopes have relatively low immunogenicity, granulocyte-macrophage colony-stimulating factor (GM-CSF) was applied to achieve efficient vaccination in the study. Among 48 patients with pancreatic cancer (10 surgically resected and 38 with advanced disease), vaccination of mutant K-ras peptides in combination with GMCSF resulted in immune responses and prolonged survival [50]. Moreover, another group also reported that vaccination of 24 patients with resected pancreatic cancer with K-ras peptide in combination with GM-CSF proved to be safe without tumor regression and median recurrencefree survival time was 8.6 months and median overall survival time was 20.3 months [51]. In one clinical study vaccination with mutated K-ras resulted in 20% long term survivors [52]. A randomized phase II placebo controlled trial using recombinant mutated K-ras protein for vaccination in combination with gemcitabine, in patients with resected pancreatic cancer, is currently ongoing [53]. Lisiansky et al. selectively kill Ras-transformed cells by over expressing the pro-apoptotic protein, p53 upregulated modulator of apoptosis (PUMA) under a Rasresponsive promoter, and assess it may become a useful, effective and safe approach to selectively target Ras-mutated tumor cells [54].

Survivin vaccines:
Survivin is a member of the inhibitor apoptosis family, which is highly upregulated in most malignancies, including pancreatic cancer [55]. In a study, murine pancreatic and lymphoma models, the survivin DNA vaccine showed significant slow tumor growth and longer survival compared with those vaccinated with vector DNA [56]. In a study, a survivin-derived peptide (AYACNTSTL) was used in combination with IFN α to vaccinate six patients who had advanced pancreatic cancers. Tetramer and enzymelinked immunosorbent spot (ELISPOT) assays revealed that more than half of the patients had manifested immunological responses to   vaccination, which were often accompanied by clinical benefits [57]. However, this vaccine still needs to be tested in a large population.

Gastrin vaccines:
Gastrin and cholecystokinin B receptor (CCKBR, also known as CCK-2) are upregulated and co-expressed in both pancreatic cell lines and human PDA specimens and have been implicated in an autocrine, paracrine, and endocrine growth pathway [58]. In a randomized, double-blind, placebo-controlled, group-sequential multicenter trial of G17DT in patients with advanced pancreatic cancer unsuitable for or unwilling to take chemotherapy, resulted in a nearly 2-fold increase in median overall survival, as compared with placebo (151 vs. 82 d, respectively; p=0.03). Anti-gastrin immune responses were noted in 73.8% of the patients and correlated with longer overall median survival versus non responders and placebo (176 vs. 63 vs. 83 days respectively, p=0.003) [59]. Gastrinbased vaccines appear therefore to be well tolerated by and could represent a new therapeutic option for pancreatic cancer.

HSP-peptide complex vaccines:
Heat shock protein (HSP), a component of HSP-peptide complex (PC), works as a peptide chaperone for stabilizing and delivering peptides. HSP-peptide complexes can be presented on MHC class I molecules on the cell surface. Tumor-derived HSP-peptide complexes have been shown to induce antitumor immune responses in preclinical studies. HSP96peptide complexes produced from resected tumor tissues were the first to be employed in anticancer vaccines. A phase I pilot trial of patients with resected pancreatic cancer who received no adjuvant radiation or chemotherapy showed the feasibility of preparing HSPCC-96 from the resected tumor. A total of 10 patients were vaccinated with 5 μg of autologous HSPPC-96 weekly for four doses. No dose limiting toxicities were observed. There was no correlation between survival and immune response, exhibiting a median overall survival of 2.2 years. Three of 10 patients were alive without disease at 2.6, 2.7 and 5-years follow up [60]. This study showed that vaccine preparation from resected tumor and the administration were feasible. Further studies need to evaluate the clinical efficacy of HSP vaccines in patients with pancreatic cancer.

WT1, cancer-testis antigens (CT) and VEGFRs vaccines:
Wilms tumor gene (WT1) protein is an attractive target for cancer immunotherapy, in a study of 32 patients with advanced pancreatic cancer was treated with WT1 vaccine in combination with gemcitabine and was well tolerated. The association between longer survival and positive delayed-type hypersensitivity to WT1 peptide was statistically significant, and longer survivors featured a higher frequency of memory-phenotype WT1-specific cytotoxic T lymphocytes both before and after treatment [61]. Median survival time and 1-year survival rate were 8.1 months and 29% respectively.
The cancer-testis (CT) antigens are expressed by tumors of different histological types at varying frequencies (10-40%) [62]. CT antigens are absent in normal somatic cells in humans and rodents but expressed only in male germ cells (such as spermatogonial stem cells, spermatogonia, spermatocytes, spermatids and spermatozoa) during spermatogenesis in the testis (but not Sertoli and/or Leydig cells) [63][64][65][66][67][68]. Fifty two percentage of the analyzed pancreatic cancer tissue expressed at least one CT antigen [69]. Recently, in a Phase I clinical trial for advanced pancreatic cancer to investigate the safety, immunostimulatory effects, and antineoplastic activity of a multi-target vaccine composed of four distinct peptides derived from cancer-testis (CT) antigens and vascular endothelial growth factor receptors (VeGFrs) was well-tolerated, and no grade 3 or 4 adverse were observed [70]. The median overall survival (OS) of this cohort was 207 days.
Two vascular endothelial growth factor receptors (VeGFrs) peptide vaccine studies using VEGF-R1 and VEGF-R2 are completed but have not reported results yet [71].
6. Telomerase reverse transcriptase (hTERT) and Her2/neu vaccines: Telomerase reverse transcriptase (hTERT) is a highly immunogenic antigen and has been the target in several vaccination studies. A small phase I/II study in patients with pancreatic cancer showed T cell responses in 63% of vaccinated patients, and prolonged survival in patients exhibiting T cell responses [72]. However, a large phase III study failed to show a survival benefit in pancreatic cancer [73]. Another study is evaluating radiation therapy, tadalafil, sargramostim, gemcitabine and telomerase vaccine (GV1001) in patients with unresectable pancreatic cancer and is currently active and waiting for result [74]. Recently, vaccination against Her2/neu is also being tested in a phase I study in patients with pancreatic cancer [75].

KIF20A derived vaccine:
Kinesin-like protein KIF20A is a protein that is encoded by the KIF20A gene, KIF20A has been shown to interact with Ras-related protein-RAB6A [76][77][78][79]. In a phase I/II trial, HLA-A24-restricted peptide vaccine derived from KIF20A was used for a patient with advanced PC, the trial came to the conclusion that the patients vaccinated with KIF20A-66 peptide had better prognosis than the control group with best supportive care, and concluded that KIF20A-66 vaccination is significantly effective as an immunotherapy against advanced PC [80]. In another phase I clinical trial, KIF20Aderived peptide vaccine with gemcitabine were analyzed and suggested that combination therapy may be feasible and promising for advanced PC [81].

B. Recombinant vaccines
Recombinant vaccines contain bacterial and viral antigen carriers thereby increasing DC activation and improving antigen presentation. They stimulate the innate immune response while efficiently recruiting and activating DCs. The most common carriers include Bacillus Calmette-Guerin (BCG), Listeria monocytogenes (LM), Salmonella and Poxviruses.

Carcinoembryonic antigen (CEA) and mucin 1 (MUC-1):
The carcinoembryonic antigen (CEA) is an oncofetal antigen that is expressed highly in the majority of pancreatic cancers [82] and MUC-1 is a membrane bound glycoprotein known to promote pancreatic cancer epithelial to mesenchymal transition and invasiveness. It also induces CD8 T cell responses and the production of anti-MUC antibodies are associated with improved survival [83]. TRICOM is a poxvirus-based vaccine containing a combination of three T-cell co-stimulatory molecules: B7-1, intercellular adhesion molecule 1 (ICAM-1) and leukocyte function associated antigen 3 (LFA-3). In a phase I study of TRICOM with CEA vaccine was conducted in 58 patients with metastatic pancreatic cancer by Marshall and colleagues. In this study, the CEA-TRICOM vaccine was used with or without GM-CSF and only one patient had pancreatic cancer. This patient had progressed with increasing pain and CA 19-9 on previous CEA vaccination. After two vaccinations with CEA-TRICOM, CA 19-9 and pain levels decreased for almost 1 year [84]. This study showed that CEA-TRICOM vaccines were safe and generated significant CEAspecific immune response with associated clinical benefit. In a study, yeast-CEA vaccine has been shown to activate DCs when used in murine models [85]. Further, Remondo et al. also showed that Yeast-CEA activates human DCs, which then successfully activated CEAspecific T-cell responses. The CEA-specific T-cell lines were able to lyse human CEA+ pancreatic tumor lines [86]. In a large randomized phase-III clinical trial of 255 patients, vaccination with a MUC-1 and CEA expressing viral vector showed no overall survival benefits [87]. Interestingly, in a study MUC1-based vaccine in combination with a celecoxib and low-dose gemcitabine was effective in preventing the progression of preneoplastic intraepithelial lesions to invasive PC, further the combination treatment elicited robust antitumor cellular and humoral immune responses and was associated with increased apoptosis in the tumor [88].

Listeria vaccines:
Listeria monocytogenes (Lm) -based vaccines stimulate both innate and adaptive immunity. In one recent study, patients bearing hepatic metastases from mesothelioma, ovarian cancer, non-small cell lung carcinoma and PC were given this L. monocytogenes strain further engineered to express mesothelin, a cell surface molecule overexpressed by a large majority of PC, mesotheliomas, ovarian cancers, and non-small cell lung carcinomas [89]. Thirty-seven percent of these patients survived 15 months or more. Half of them patients were those harboring metastatic PC, and immunological analyses revealed that they had developed listeriolysin O-and mesothelin-specific T-cell responses. While a mesothelin vaccine, using genetically modified live attenuated listeria as a vector for the antigen has also entered clinical trial [90].

C. Antigen pulsed dendritic cell (DCs) vaccines
DCs are the most potent antigen presenting cells (APCs) that are capable of priming naïve T cells and can stimulate memory T cells and B cells to generate antigen specific response. Antigen pulsed DCs is another vaccination strategy where patient DCs are isolated, pulsed with peptides, autologous, or allogeneic tumor lysate, or transfected with RNA, and injected back to the patients. In a study of 12 patients (10 with pancreatic cancer), where MUC-1 pulsed DCs were given as adjuvant therapy following resection, 4 of the 12 patients were alive at 4 years [91]. In the second study, a DC-based vaccine alone or combined with Lymphokine activated killer (LAK) cells was administered together with gemcitabine and/or S-1 to 49 patients with advanced pancreatic adenocarcinoma [92]. Median survival in these patients was 360 days. Patients receiving DC vaccine along with chemotherapy and LAK cell therapy had prolonged survival compared with patients who received DC vaccine and chemotherapy. Of all 49 patients, 2 had complete remission, 5 had partial remission and 10 had stable disease. Thus, the study concluded that DC-based vaccine therapy with chemotherapy was shown to be safe and may induce responses.

D. Whole tumor cell vaccines
Whole tumor cell vaccines are another strategy that has shown promise in pancreatic cancer. Autologous whole tumor cells remain a potent vehicle for generating antitumor immunity. This is because tumor cells express all relevant candidate TAAs, including both known and unidentified. Two allogeneic whole cell based anticancer vaccines are currently being investigated for their safety and antineoplastic effects in PC patients.
a. Algenpantucel-L: Algenpantucel-L is the most clinically advanced and promising immunotherapy for pancreatic cancer. Algenpantucel-L (also known as hyperacute-pancreatic cancer vaccine) consists an 2 irradiated, live, human allogeneic pancreatic cancer cell lines that express murine α-1,3-galactosyltransferase, which is responsible for the synthesis of α-galactosylated epitopes on cell surface proteins. Hardacre and colleagues presented the results of an open-label, multi-institutional Phase II clinical trial investigating Algenpantucel-L in combination with standard adjuvant chemoradiotherapy for the treatment of resected PC patients [93]. In this study 69 out of 73 patients were evaluable and they received 100 million cells (N=43) or 300 million cells (N= 26) injected intradermally in up to 14 vaccinations. No serious adverse events were attributed to the immunotherapy. After a median follow-up of 21 months, the 12-month disease-free survival was 62%, and the 12-month overall survival was 86%. The most common adverse events were injection site pain and induration. The study concluded that the addition of algenpantucel-L to standard adjuvant therapy for resected pancreatic cancer may improve survival. A multi-institutional, phase 3 study is ongoing (ClinicalTrials.gov identifier, NCT01072981) [94].
b. Allogeneic GM-CSF vaccine therapy: Allogeneic Granulocytemacrophage colony-stimulating factor (GM-CSF) vaccine therapy, called GVAX, has been tested in a variety of early phase clinical trials. In a phase I clinical study, tumor cells, which were modified to express the immunomodulating cytokine GM-CSF, were given to 14 patients [95]. Three patients had delayed-type hypersensitivity responses to autologous tumor cells and those 3 patients had a longer disease free survival. The Latter, in a phase-II study with a similar approach, 60 patients with resected pancreatic cancer were treated, yielding a disease free survival of 17.3 months and an overall survival of 24.8 months [96]. While the results did not superior result of previous study, other studies using similar approaches, or combining whole tumor vaccination with cyclophosphamide alone, or with conventional chemotherapy, are ongoing [97][98][99][100].

Challenges and Enhancing Vaccine Efficacy by Combinatorial therapy
The effective vaccine also should seek to provide long term memory to prevent tumor recurrence [40]. Scientists believe that for total tumor elimination, both the innate and adaptive immune systems should be activated. In current status, immune modulation and immunosuppressive environment by cancer seems to be the major concern and the main challenge for vaccine therapy. One of the means that PC cells evade destruction is through "antigen loss variance". In a study, Jansen et al. found that tumor recurrence is associated with tumor depigmentation and antigen loss which occurs in immunoediting. However, their studies suggest that even with reduced expression these tumors are still susceptible to antigen-specific antitumor vaccine. Further, their study also demonstrated that targeting Treg and down modulation or removal of the Treg population prior to and/or during immunotherapy may make these cells less susceptible to antigen loss [101].
A tumor can have many different types of cells in it, each with different cell-surface antigens. Vaccine targeting multiple antigens presented by a tumor cell may be advantageous and can enhance vaccine efficacy, but there is a risk of autoimmunity. Though, antigen specific-antitumor vaccine can decrease the risk of autoimmunity but because the immune response is directed to a single epitope, tumors can evade destruction through an antigen loss variance. This also raises important questions regarding antigen-specific immunization approaches. There are many data supporting that "epitope spreading" may reduce antigen loss variance, as sometimes an immune response to a single antigen will lead to the development of immunity against other antigens on the same tumor. Thus, epitope spreading in this context has obvious important implications for the design of antigen-specific antitumor immunotherapies [102].
As stated earlier, breaking down the immunosuppressive environment of cancer is another need of vaccine therapy for optimization of its efficacy, many researchers has suggested that the efficacy of vaccine therapy can be enhanced by combinatorial therapy. It has been found that cytotoxic T lymphocyte-associated antigen 4 (CTLA4) and PD1 are two well known co-inhibitory molecules that decreases antitumor responses [103]. In a small clinical trial GM-CSF vaccine along with CTLA4 blockade found increased inflammatory infiltrated and tumor regression [104]. Similarly, in a preclinical study of murine model vaccine was given in combination with PD1 blockade, mice receiving combination therapy had increased overall survival and decreased tumor growth [105]. In one study, it was found that animals treated with vaccine along with antibodies to CTLA4 and PD1 have significantly higher overall survival compared with animals treated with vaccine and either antibody alone, suggesting that combining blockade of multiple inhibitory pathway decreases T cell anergy and improves T cell responsiveness [106]. Many of the preclinical studies have demonstrated that some small molecules targeted therapeutics in combination with vaccine have the ability to enhance vaccine mediated T cell lysis of tumors. Small molecule inhibitors such as BCL-2 inhibitor, tyrosine kinase inhibitor sunitinib and B-raf inhibitor have been shown to enhance the ratio and expression of TAA-specific T cells to regulatory cells, resulting in enhanced vaccine efficacy [107][108][109][110][111]. Further, radiation therapy (RT) may act synergistically with vaccine therapy to enhance immune responses, inhibit immunosuppression, induction of DNA damage, and/or alter the phenotype of tumor cells, thus rendering them more susceptible to immune-mediated killing [112,113]. It has been found that RT bears the potential to stimulate DC medicated antitumor immunity in this way [112]. Further, it has also been found that the destruction of even a small percentage of tumor cells by radiation could result in cross-priming and presentation of tumor antigens to the immune system, thereby potentiating antitumor responses [114]. The challenge for now is how to exploit radiationinduced changes in tumor-cell antigens and how to induce effective immune responses to these cumulatively immunogenic stimuli.
Recently, Vaccine has also been used with other combinatorial therapies like hormonal, monoclonal antibodies and chemotherapy; many of these preclinical and clinical studies have shown potential benefits over the vaccine therapy alone.

Logical Problems for Vaccine Therapy
Most clinical trials investigating a cancer vaccine have failed or had very modest responses, possible because of the following reasons: It is hard to treat advanced disease stage or bulky tumor with vaccines, because bulky tumor actively suppresses the immune system. The most suitable stage for a cancer vaccine is likely to be early disease or after de-bulking of the tumor by surgery, chemotherapy or by radiotherapy [37]. Antigen specific anti-tumor vaccines are more at risk for antigen loss variance. Cancer vaccines that target just one tumor antigen are likely to be less effective. The most effective cancer vaccine is likely to raise an immune response against multiple tumor antigens to minimize the chance of the tumor being able to mutate and become resistant to the therapy [102]. But vaccines targeting multiple tumor antigens may increase the risk of autoimmunity [115]. Numerous clinical trials carried out in the past have treated patients who have received numerous cycles of chemotherapy. As chemotherapy, is often myelosuppressive and immunosuppressive. So, there is little point of giving a cancer vaccine to a patient who is immune suppressed. It may take some times for vaccine to show an immune response and thus, vaccine may not be suitable for the highly malignant tumors like pancreatic cancer that can produce marked clinical deterioration, or even death, within this time period.

Perspectives
Although vaccine therapy as a single agent has encouraging results, clinical trials in PC patients have been underwhelming and disappointing. Most of these clinical studies identified a number of critical aspects that must be carefully considered in the design the next generation of cancer vaccines. As stated earlier dysfunctional immune system in PC further give rise to tumor induced immunosuppressive environment has a critical role in the development and progression of tumor. Which consist of not only cancer cells, but also immune suppressive cells (CAFs, tolerogenic DCs, MDSCs, TAMs and Treg cells) and cancer cells-derived soluble factors that promote the induction of tolerance. So, the novel vaccine therapy strategy should be designed for breaking immunosuppression within the tumor microenvironment, to inhibit immunologic checkpoint blockade and to modulate tumor microenvironment. Thus, this may require a combinatorial therapeutic approach which includes chemotherapy, radiation, surgery and immunotherapy.