【论文】WT1靶向癌症疫苗可延长胰腺癌患者的生存期
Wilms’ tumor 1 (WT1)-targeted cancer vaccines to extend survival for patients with pancreatic cancer
Shigeo Koido*,1,2, Masato Okamoto3, Shigetaka Shimodaira4 & Haruo Sugiyama5
1Division of Gastroenterology & Hepatology, Department of Internal Medicine, The Jikei University School of Medicine, Kashiwa Hospital, Kashiwa City, Chiba 277-8567, Japan
2Institute of Clinical Medicine & Research, The Jikei University School of Medicine, Kashiwa City, Chiba 277-8567, Japan
3Department of Advanced Immunotherapeutics, Kitasato University School of Pharmacy, Tokyo 108-8641, Japan
4Cell Processing Center, Shinshu University Hospital, Nagano 390-8621, Japan
5Department of Functional Diagnostic Science, Graduate School of Medicine, Osaka University, Suita City, Osaka 565- 0871, Japan
Keywords: cancer vaccine • dendritic cell • pancreatic cancer • peptide • WT1
Despite novel chemotherapy treatments, pancreatic ductal adenocarcinoma (PDA) remains a lethal disease. New targeted cancer vaccines may represent a viable option for patients with PDA. The Wilms’ tumor 1 (WT1) antigen is one of the most widely expressed tumor-associated antigens in various types of tumors, including PDA. Recent reports have indicated that WT1-targeted cancer vaccines for patients with PDA mediated a potent antitumor effect when combined with chemotherapy in preclinical and clinical studies. This review summarizes the early-phase clinical trials of WT1-targeted cancer vaccines (peptide vaccines and dendritic cell-based vaccines) for PDA. Moreover, we will discuss future strategies for PDA treatments using WT1- specific cancer vaccines combined with immune checkpoint therapies to maximize the clinical effectiveness of PDA treatments.
Pancreatic cancer
The vast majority (more than 95%) of pancreatic tumors arise in the exocrine component of the pancreas, and of these, the most common is ductal adenocarcinoma (PDA) [1]. Patients with PDA have an extremely poor prognosis, with an overall 5-year survival rate of less than 5% and a median survival time (MST) of 4–6 months [2]. Surgical resection (pancreaticoduodenectomy) is an option for only 10% of PDA patients. The majority (approximately 80%) of PDA patients present with locally advanced or metastatic disease, which precludes surgery. These advanced PDA patients have an extremely poor prognosis. The chemotherapeutic agent gemcitabine was once considered a standard of care for patients with PDA. This agent improved the median overall survival (OS); however, the treated PDA patients usually did not survive longer than 6 months [3]. New chemotherapeutic regimes, such as FOLFIRINOX (oxaliplatin, irinotecan,eucovorin and 5-fluorouracil [5-FU]) [4] and gemcitabine/human-albumin-bound paclitaxel (nab-PTX) [5] significantly improved outcomes for advanced PDA compared with gemcitabine alone; however, such a lethal disease still poses significant challenges for PDA patients. Moreover, FOLFIRINOX is suitable for only PDA patients with a good performance status because of its significant chemotoxicity. Even if patients receive surgical resection after early cancer detection, local recurrence and multiple liver metastases occur in the majority of PDA patients. Therefore, effective adjuvants, such as S-1, an oral 5-FU, are also required to improve clinical outcomes [6,7]. Although treatment with both chemotherapy and radiation therapy has been extensively investigated, the current cytotoxic and targeted therapies have provided only limited success thus far for PDA, which remains a lethal disease. Therefore, the development of new therapeutic strategies for extending the survival of the vast majority of PDA patients has traditionally been considered particularly challenging. Importantly, PDA cells express multiple tumor-associated antigens (TAAs), such as Wilms’ tumor 1 (WT1) [8], mucin 1 (MUC1) [9], human telomerase reverse transcriptase (hTERT) [10], mutated K-RAS [11], survivin [12], carcinoembryonic antigen (CEA) [13], epidermal growth factor receptor 2 (HER-2) [14] and p53 [15]. PDA that develops chemoresistance expresses TAAs; thus, it is still a suitable target for cancer vaccines. Therefore, combination therapy consisting of cancer vaccines and conventional therapy may be an attractive treatment for PDA. Combination therapy can also be used in PDA patients in remission to prevent local recurrence and metastasis after surgical resection.
Pancreatic cancer microenvironment
PDA is one of the most stroma-rich and hypovascular cancers [16,17]. The PDA stroma induces resistance to both chemotherapy and radiation [18] and constitutes a barrier to the delivery of therapeutic agents to PDA cells [19]. Therefore, the factors associated with conventional therapy failure may involve PDA microenvironment characteristics. The dense fibrous stroma in PDA is very heterogeneous and consists of an abundant extracellular matrix (e.g., collagen, hyaluronan, fibrinogen and fibrin), other soluble proteins (e.g., cytokines and growth factors) and nonepithelial cells (e.g., fibroblasts, myofibroblasts, inflammatory cells, stellate cells, blood vessels, endothelial cells, pericytes and nerves) [20]. Moreover, cancer-associated fibroblasts, tolerogenic dendritic cells (DCs), myeloidderived suppressor cells (MDSCs), immunosuppressive tumor-associated macrophages, tumor-associated neutrophils and regulatory T cells (Tregs) in stroma suppress antitumor immunity and promote PDA cell invasiveness through the production of immunosuppressive cytokines, such as interleukin-10 (IL-10) and tumor growth factor-β (TGF-β), along with depletion of arginine and the augmentation of reactive oxygen species and nitrogen oxide [21]. Although TAA-targeted cancer vaccines have been shown to be promising strategies in preclinical studies, the abundance of immunosuppressive cells in the PDA stroma leads to low antigen-specific cytotoxic T lymphocyte (CTL) infiltration into PDA cells [22]; therefore, the ability of TAA-targeted cancer vaccines alone to eliminate large established PDA may be limited. Indeed, the heterogeneous nature of the PDA stroma promotes tumorigenesis, leading to limited antitumor cytotoxicity and is thus associated with poor prognoses [23]. PDA stroma and cells promote immunosuppression and are potential therapeutic targets of cancer vaccines in patients with advanced PDA. Specific strategies for targeting regulatory immune cells (e.g., MDSCs, tumor-associated macrophages or Tregs) and stromal cells (e.g., cancer-associated fibroblasts or stellate cells) using anti-CD40 mAb, anti-CTLA-4 mAb, anti-PDL1, anti-PD-1, anti-CSF-1R mAb and RLR ligand PDE5 inhibitors have been reported to be effective immune-based therapies in preclinical studies [24]. Thus far, only human epidermal growth factor receptor type 1 (HER1/EGFR) inhibitor in combination with gemcitabine has significantly improved the survival of PDA patients in a randomized Phase III trial [25]. In contrast to immunosuppressive cells, the numbers of CD4+ and CD8+ effector T cells and natural killer (NK) cells are minimal in the PDA microenvironment, resulting in nonimmunogenic PDA [22,26]. Indeed, the PDA microenvironment of surgical specimens is infiltrated with high numbers of immunosuppressive cells and low numbers of CTLs [26]. Moreover, tumorinfiltrating CD4+ and CD8+ T cells have exhibited nonfunctional phenotypes [22,26]. Cross-talk between PDA cells and their microenvironment is very complicated and drives tumor progression. Recent studies have indicated that immune checkpoint molecules, such as CTL-associated antigen 4 (CTLA-4) and programmed death-1 (PD-1), transduce inhibitory signals during T-cell activation [27]. Moreover, tumor cells express their ligands, such as programmed death receptor ligand 1 (PD-L1), which results in the inhibition of antitumor immune responses. Upregulation of CTLA- 4, PD-1 and PD-L1 has been associated with poor prognoses[27,28]. Characterizing the underlying molecular mechanism for the cross-talk between PDA cells and their microenvironment has been challenging [29].
The WT1 antigen
The WT1 gene was originally defined as a tumor suppressor gene involved in the etiology of Wilms’ tumor [30]; however, it also manifests oncogenic activities[31–33]. The WT1 gene encodes a zinc finger transcription factor, WT1, which has been identified as a potent transcriptional regulator that correlates with cell development and progression in various cancer types [32,34,35]. Indeed, WT1 mRNA is overexpressed in various types of solid tumors (e.g., lung, breast and colon tumors, gastrointestinal stromal tumors and pancreatic cancer) and in leukemia and myelodysplastic syndrome [8,36,37]. Importantly, WT1 has been shown to activate transcription of the IL-10 gene, thereby possibly promoting tumor escape from immune surveillance[38]. After patients with WT1-expressing tumors undergo treatment, they spontaneously produce WT1- specific IgM- and IgG-type antibodies and CTLs or helper T cells against WT1 antigens, suggesting that WT1 is highly immunogenic and a promising targeted antigen for cancer vaccines [32,39–41]. Moreover, WT1 plays an essential role in maintaining the transformation of cancers, and tumor escape from immune surveillance as a result of its downmodulation is unlikely to occur [42]. Thus, WT1 may serve as a diagnosis predictor and solid tumor prognosis marker in patients who have received cancer vaccines [43–45]. Recently, WT1 was ranked as the top antigen in a list of 75 TAAs by a National Cancer Institute prioritization project based on several factors: therapeutic function, immunogenicity, the role of the antigen in oncogenicity, specificity, the expression level and percentage of antigen-positive cells, stem cell expression, the number of patients with antigen-positive cancers, the number of antigenic epitopes and the cellular location of antigen expression [46]. Moreover, 75% of PDA cells express WT1 protein [8]. These findings suggest that a new era of WT1-targeted cancer vaccines for PDA patients may be imminent.
WT1-targeting chemoimmunotherapy for PDA cells
As chemotherapeutic agents interfere with DNA synthesis and replication, these agents are cytotoxic to peripheral lymphocytes and tumor cells. Therefore, it has been well accepted that chemotherapy blunts antitumor immune responses. However, some chemotherapeutic agents and molecular targeted agents can induce immunogenic tumor cell death (necrosis or apoptosis), which can lead to immature DC phagocytosis and DC activation. This pathway results in processing acceleration and antigenic peptide presentation to T cells, followed by the induction of antitumor immune responses without Treg induction [47–49]. For example, only tumor cells that undergo immunogenic apoptosis ectopically produce various danger signals, such as high mobility group protein box 1, heat shock proteins 70/90, adenosine triphosphate and calreticulin (CRT), a Ca2+-binding chaperone that is found in different apoptotic stages and promotes TAAs to traffic to the antigen-presenting compartment in DCs [49–51]. Moreover, heat shock proteins 70 and high mobility group protein box 1 that are passively released from necrotic tumor cells interact with Toll-like receptor 4 on DCs, and they activate antigen processing and presentation machinery in DCs [52–54]. Our recent report also indicated that human cholangiocarcinoma produced CRT upon treatment with gemcitabine [55]. Some chemotherapeutic agents and molecular targeted agents can also induce T-cell activation, deplete suppressive immune cells, such as Tregs and MDSCs, and enhance tumor cell susceptibility to CTL-mediated lysis [48]. As a result, a positive interaction between cancer vaccines and chemotherapy may herald a new era for cancer immunotherapy. Interestingly, we also found that treatment of some human PDA cell lines with standard PDA cytotoxic agents, such as gemcitabine or 5-FU, resulted in enhanced WT1 mRNA levels [56]. Human PDA cell treatment with these chemotherapeutic agents also resulted in a WT1 protein shift from the nucleus to the cytoplasm, which promoted proteasomal processing of WT1 protein and presentation of WT1 peptide in major histocompatibility complex (MHC) class I molecules on human PDA cells. Certain TAAs, such as WT1, which usually exhibit low levels on PDA cells, might be uncovered by treatment with various chemotherapeutic agents. A recent report indicated that these uncovered TAAs are good targets for cancer vaccines because they can be effectively recognized by antigen-specific CTLs when the TAAs are expressed well [57]. Indeed, WT1- specific CTLs lysed human PDA cells treated with an optimal dose of gemcitabine more efficiently than untreated PDA cells [56]. Therefore, some chemotherapeutic agents can enhance WT1 peptide presentation in human PDA cells and can sensitize the PDA cells to the cytotoxic effects of WT1-specific CTLs. Additionally, our recent reports indicate that the combination therapy of trastuzumab conjugated to a cytotoxic agent (T-DM1) with gemcitabine may be a promising treatment for PDA cells [58] or breast cancer cells [59] with low HER2 expression levels as a result of the unique HER2-upregulating effect of gemcitabine. Importantly, cancer patients who have previously received cancer vaccines could also have more clinical benefits than nonvaccinated patients [60]. Increasing evidence has suggested that chemotherapy-induced immunogenic modulation of PDA cells may enhance antigen processing and calreticulin exposure, resulting in enhanced CTL responses. WT1-targeting cancer vaccines may possibly achieve better success when used in combination with chemotherapy [48,61]. Additionally, WT1-specific CTLs that are induced by cancer vaccines might kill PDA cells that have already acquired resistance.
WT1-targeting chemoimmunotherapy for cancer stem cells
Chemotherapy and radiation therapies that rapidly target proliferating cells can eradicate certain tumor cells. In tumor cells, however, there is a small population of cancer-initiating/cancer stem cells (CSCs) that have the capacity for self-renewal, differentiation and tumor regeneration [62]. CSCs that are resistant to chemoradiation therapy frequently lead to uncontrolled amplification of differentiated cancer cell populations, resulting in therapeutic failure and cancer recurrence[63]. CSCs may be the culprits behind cancer recurrence and metastasis after standard treatment. Therefore, the eradication of chemotherapy- and/ or radiation therapy-resistant CSC fractions may be critical for prognosis improvements in PDA patients. CSC-associated antigens are generally classified into two types: CSC-specific antigens, such as CD44 [64], SOX2, POU5F1, LGR5 and ALDH1A1 [65]; and shared antigens between CSCs and more differentiated cancer cells, such as CEP55 [66], MUC1 [67,68]and WT1 [69–72]. Therefore, these remaining CSCs are still attractive targets for cancer vaccines [68,73]. It is desirable to develop a novel combination therapy that consists of CSC-targeting cancer vaccines and conventional therapies (e.g., surgery, chemotherapy and radiation therapy) to target the bulk of cancer cells. For example, DC-based cancer vaccines [74], γδ T cells [75]and NK cells [76] have the capacity to eliminate human CSCs in vitro. We have also reported that CSC-loaded DCs induced proliferation of CD4+ and CD8+ T cells that produced interferon (IFN)-γ and killed CSCs in a murine study [68]. As CSCs express WT1 [69–72], the development of surgery/chemotherapy/radiation therapies combined with WT1-targeting cancer vaccines might be highly desirable.
WT1-targeting vaccines in clinical trials
Recently, MHC class I-restricted CTL and class IIrestricted helper epitopes of WT1 antigens were identified and used in clinical trials without significant adverse effects (Table 1) [77].
Clinical trials of WT1-targeted cancer vaccines for patients with PDA have been performed. Thus far, 6 phase I trials of WT1-targeted cancer vaccine and standard chemotherapy combination therapies for advanced PDA patients have been published (Table 2).
The WT1-targeted cancer vaccines in clinical trials can be classified into three types: MHC class I-restricted WT1 (WT1-I) peptide vaccines[43,78], WT1-I peptide-pulsed DCs (DC/WT1- I) [79–81] and MHC class I- and class II-restricted WT1 peptide (WT1-I/II)-pulsed DCs (DC/WT1-I/II) [82].
WT1-I peptide vaccines & chemotherapy
WT1-peptide vaccines are frequently used in clinical trials because they are safe and economical. In a Phase I study for PDA patients, a WT1-I peptide vaccine was combined with gemcitabine [43,78]. Twentyfive patients (nine advanced with PDA, eight with gallbladder cancer, four with intrahepatic cancer and four with extrahepatic bile duct cancer) received WT1 peptide restricted with human leukocyte antigen (HLA)-A*02:01, -A*02:06 and/or -A*24:02 emulsified in Montanide ISA51 adjuvant in combination with gemcitabine [78]. In this trial, the adverse events were comparable with those for gemcitabine alone. After the therapy, WT1 peptide-specific delayed-type hypersensitivity (DTH) was detected in two patients. By tetramer assay, WT1-specific T-cell responses in peripheral blood mononuclear cells (PBMCs) were observed in 13 of 22 patients (59%), including 9 PDA patients. The disease control rate at 2 months was 89% for PDA and 50% for biliary tract cancer. The MST for pancreatic cancer was 259 days. In this trial, the objective clinical efficacy was not apparent. Additionally, the chemoimmunotherapy was well tolerated. We also conducted a combination therapy of WT1 peptide restricted with HLA-A*24:02 emulsified in Montanide ISA51 adjuvant and gemcitabine [43]. Thirtytwo HLA-A*24:02+ patients with PDA were enrolled in this Phase 1 study. This combination therapy was also well tolerated. The MST and 1-year survival rates were 8.1 months and 29%, respectively. Interestingly, the longer survival times were significantly associated with WT1-specific DTH-positive reactions and a high percentage of memory-phenotype WT1-specific CTLs in PBMCs both before and after treatment. Therefore, both WT1-specific DTH reactions and memory-phenotype WT1-specific CTLs may serve as useful prognostic markers of PDA patient survival following chemoimmunotherapy. In this trial, a 44-yearold male with locally advanced pancreatic head cancer (T4N1M0; stage III) showed >80% regression of the primary lesion after 5 months of treatment and underwent a complete surgical resection. Histopathological analysis of his specimen showed mononuclear cell infiltration around the ductal adenocarcinoma with fibrotic changes. This patient demonstrated a WT1-specific DTH reaction and a high percentage of memory-phenotype WT1-specific CTLs over the entire treatment course. Interestingly, the percentage of WT1-CTLs of tumor-infiltrating CD3+ CD8+ T cells was 2.48%, which was approximately six-times higher than the percentage of WT1-CTLs found in PBMCs (0.39%). Therefore, this chemoimmunotherapy can be expected to induce augmented antitumor immunity in PDA patients. To determine the clinical efficacy of this chemoimmunotherapy, we have started a Phase II randomized clinical study.
DC/WT1-I vaccine & chemotherapy
DCs are the most potent antigen-presenting cells and play pivotal roles in the initiation, programming and regulation of antitumor immune responses [83,84]. Therefore, TAA-loaded DCs have been developed as cancer vaccines because of their powerful capacity to induce antigen-specific CD4+ and CD8+ CTL responses [85]. Monocyte-derived mature DCs can be easily expanded in vitro; loaded with tumor cell lysates, TAAs, mRNA or whole tumor cells; and then injected into cancer patients often in combination with chemotherapy in clinical trials. In a retrospective study, we first evaluated the clinical and immunological responses of DC-based cancer vaccines, including WT1-I pulsed DCs combined with a standard chemotherapy, gemcitabine and S-1, in 49 PDA patients [79]. The combination therapy was safe. Before the chemoimmunotherapy, 46 of the 49 patients had been treated with chemotherapy or radiotherapy without significant clinical benefits. Despite this extensive pretreatment, two patients experienced complete remission (CR), five experienced partial remission (PR) and ten had stable disease (SD) with the subsequent combination treatment. Furthermore, the MST has now reached as long as 360 days from the first vaccination, a significant prolongation compared with historical controls treated with GEM or S-1. Therefore, chemoimmunotherapy targeting TAAs, including WT1-I, may be effective in patients with PDA that is refractory to standard treatment. These authors then conducted a retrospective analysis of the chemoimmunotherapy by expanding the sample size into multiple centers [80]. In this study, 255 PDA patients who received standard chemotherapy combined with multiple peptides, including WT1-I-pulsed DC vaccines, were analyzed. The MST from diagnosis was 16.5 months, and the MST from the first vaccination was 9.9 months. Importantly, an antigen-specific DTH reaction induced by the vaccination was a treatment-related prognostic factor for better survival. These findings strongly suggest that there are good responders for chemoimmunotherapy in PDA patients [86]. These findings need to be addressed in well-controlled prospective trials. In a prospective Phase I study, 10 PDA patients were treated with a combination therapy of a DC vaccine pulsed with only MHC class I (HLA-A*24:02)-restricted WT1 peptide and gemcitabine [81]. In this study, the disease control rate and median OS were 60% (six SD and four PR) and 243 days, respectively. Moreover, PDA patients with multiple liver metastases and high C-reactive protein and IL-8 levels showed poor survival rates, even when they had successfully induced WT1-specific immune responses by vaccines. Therefore, induction of only WT1-specific CD8+ T cells by DC/WT1-I may be not sufficient to induce and maintain antitumor immunity in PDA patients with organ metastases.
DC/WT1-I/II vaccine & chemotherapy
It is well known that efficient antitumor immunity requires not only antigen-specific CD8+ but also CD4+ T cells. CD4+ T cells are widely known as T helper cells and are required for priming, generating and maintaining efficient CD8+ memory type effector T cells [87]. Moreover, CD4+ T cells provide help to CD8+ CTLs by activating DCs via CD40-CD40L interactions and/or IL-2 production [88]. Interestingly, a recent study demonstrated that a single infusion of a clonal population of NY-ESO-1 antigen-specific CD4+ T cells into a patient with refractory metastatic melanoma resulted in durable clinical remission [89]. We also reported that adoptive transfer of human MUC1-specific CD4+ T cells into human MUC1- positive tumor bearing Rag2(-/-) mice resulted in lung metastasis prevention [90]. Therefore, it is essential to activate both CD4+ and CD8+ T cells simultaneously for the induction of antitumor immunity. Based on these findings, we conducted a Phase I pilot chemoimmunotherapy study using DC/WT1-I/II vaccines and standard chemotherapy (gemcitabine and/ or S-1) in seven advanced PDA patients [77,82]. DC/ WT1-I/II vaccines were generated with mature DCs pulsed with three types of WT1 peptides restricted to HLA-A*02:01, A*02:06 (126–134: RMFPNAPYL) or A*24:02 (235–243: CYTWNQMNL), and DRB1*04:05, DRB1*08:03, DRB1*15:01, DRB1*15:02, DPB1*05:01 or DPB1*09:01 (332–347:KRYFKLSHLQMHSRKH) (Table 1). The combination therapy was well tolerated, and six of seven PDA patients exhibited SD but no CR or PR. After vaccination, WT1-specific CD4+ and CD8+ T cells that produced high IFN-γ levels were detected. WT1 peptidespecific DTH was detected in four of the seven PDA patients (three had strong DTH reactions). Moreover, the MST and the median progression-free survival times of the PDA patients who were vaccinated with DC/WT1-I/II (n = 7) were significantly longer than the MST and progression-free survival times for those receiving the DC/WT1-I (n = 2) or DC/WT1-II (n = 1) vaccines. These findings support the hypothesis that the coactivation of WT1-specific helper CD4+ T cells upon DC/WT1-I/II vaccines is capable of activating the proliferation and maintenance of functional WT1-specific CD8+ CTLs. WT1-specific CTLs induced by DC/WT1-I vaccines may be functionally impaired, leading to short-lived antitumor immune responses. Importantly, all 3 PDA patients with strong WT1-specific DTH reactions had a median OS of 717 days (582, 717 and more than 1050 days). Surprisingly, a PDA patient remained alive for more than 1050 days and received more than 71 DC/WT1-I/ II vaccinations despite the presence of multiple liver metastases [91]. These three super-responders maintained long-term WT1-specific CD4+ and CD8+ T-cell responses with low plasma IL-6 and IL-8 levels during the entire treatment period [91]. Additionally, the post-treatment neutrophil-to-lymphocyte ratio and the HLA-DR and CD83 mean fluorescence intensities on DCs were also treatment-related prognostic factors for better survival [92]. In super-responders, stable vaccination by DC/WT1-I/II continuously elicited not only WT1-specific CD4+ T cells but also WT1-specific long-lived memory and central memory type effector CD8+ T cells [77,82]. These long-lived WT1-specific effector CD8+ T-cell PBMCs patrolled and recognized PDA cells and were therefore associated with long-term SD and a clinical benefit. The response ratio of gemcitabine alone in advanced PDA patients is approximately 10%. Therefore, the long-term SD might be a unique characteristic of DC/WT1-I/II vaccines in combination with gemcitabine [77,82]. The clinical efficacy of cancer vaccines targeting the WT1 protein should be clarified in a large-scale clinical study [32].
WT1 expression in tumor vascularization
Angiogenesis is an important step in tumor growth and metastasis [93]. A recent report indicated that WT1 is expressed in not only tumor cells but also tumor endothelial cells, hematopoietic progenitor cells and MDSCs [94]. WT1 expression in tumor vascular endothelial cells was detected in 95% of 113 tumors of different origins, including PDA [94]. Moreover, WT1 regulated the transcriptional activation of ETS-1 and played a crucial role in tumor vascularization via the regulation of endothelial cell proliferation and migration [94]. Interestingly, WT1 inhibition is involved in the regression of tumor angiogenesis and enhances antitumor immune responses, which results in inhibition of tumor metastasis, regression of established tumors and a survival benefit. Thus, the multiple tasks of WT1 are not only limited to the tumor growth regulation via modulation of tumor vascularization but also involved in immune responses and metastasis formation[95,96]. In 20 examined human PDAs, all exhibited WT1-positive tumor vessels [94]. Thus far, cancer vaccines targeting only vascular endothelial cells with a VEGFR2 epitope peptide and gemcitabine were conducted in 18 PDA patients and showed no sufficient clinical benefit but had peptide-specific CTL responses [97]. However, WT1-specific CTLs may target not only PDA cells but also tumor endothelial cells and MDSCs in the tumor microenvironment, which has resulted in good clinical benefits for PDA.
Conclusion
Despite novel chemotherapy treatments, the prognosis for patients with PDA remains grim. Innovative therapies are urgently needed to extend survival for patients with PDA. Importantly, PDA cells that develop chemotherapeutic agent resistance are still suitable targets for TAA-specific cancer vaccines. WT1 is an intracellular oncogenic transcription factor widely expressed in various types of tumors, including PDA, and in pancreatic CSCs and tumor vascular endothelial cells in the PDA microenvironment. The development of a curative novel therapy is likely to require eradication of the bulk of PDA cells, CSCs and tumor vascular endothelial cells. Therefore, a combined approach of conventional therapies, such as chemotherapy or radiation therapy, that kill the bulk of PDA cells and WT1-specific memory CTLs that target PDA cells, CSCs fractions and tumor vascular endothelial cells may represent a more promising approach for the treatment of PDA patients. Longlived WT1-specific memory and central memory type effector CD8+ T cells can be efficiently induced by DCs pulsed with MHC class I- and class II-restricted multiple WT1 peptides. PDA patients who present a good immune response do not prove cause and effect without a randomized clinical trial. Because WT1-targeted immunotherapy was combined with chemotherapy in early Phase I studies, it is difficult to assess the true effect of the vaccine. Therefore, we have conducted a Phase II randomized clinical trial, and the results will be reported soon (UMIN000005248).
Future perspective
Effective cancer vaccines that selectively target PDA cells, CSCs and stroma cells with little toxicity in combination with conventional chemotherapies are urgently needed for patients with advanced PDA. PD-1 is highly expressed by antigen-specific CTLs and is associated with impaired CTL function. However, PD-L1 is not constitutively expressed but is induced in response to IFN-γ predominantly produced by CTLs in some tumors. Inhibiting PD1/PD-L1 signaling may induce a survival benefit in PDA patients. Antibodies can be used to block inhibitory ligand:receptor interactions by acting on tumor cells, DCs (i.e., anti-PD-L1) or T cells (e.g., anti-PD-1 or anti-CTLA-4) [98]. Combining the blockade of multiple inhibitory pathways synergistically decreases T-cell anergy and improves T-cell responsiveness against tumors [99]. PDA is considered a nonimmunogenic tumor, at least in the portion of the tumor microenvironment that provides a formidable barrier to T-cell infiltration [25]. However, efficient cancer vaccines generated with granulocyte–macrophage colonystimulating factor-secreting, allogeneic PDA cell vaccines (GVAX) induced T-cell infiltration and resulted in PD-1 upregulation in T cells and PD-L1 in PDA cells [26]. Therefore, activated T-cell infiltration in the PDA microenvironment has been induced by efficient cancer vaccines and has resulted in immunogenic PDA [26,100]. Immune checkpoint blockade has not been very effective in vaccine-naïve PDA to date. However, recent results suggest that patients with vaccine-primed PDA might be better candidates for immune checkpoint therapies than vaccine-naïve patients. Therefore, combining cancer vaccines based on WT1-specific CTLs and DC/WT1-I/II cells [77,82] and antibodies against proteins, such as PD-1, CTLA-4 and PD-L1, may promote therapeutic synergy and long-term antitumor immunity. PDA patients who receive DC/WT1-I/II vaccines may be better candidates for immune checkpoint-targeting therapies; therefore, these combination therapies may be future options.
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