Immunotherapy in pancreatic cancer treatment: a new frontier

Abstract

Pancreatic cancer is a highly aggressive and lethal cancer characterized by high invasiveness, local and extensive dissemination at time of diagnosis and resistance to treatment. Few therapies have shown efficacy in the past and even standard of care therapies yield only modest improvements in the mortality of patients with advanced or metastatic disease. Efforts have been undertaken to study the pancreatic tumor microenvironment and have established its complex and immunosuppressive nature which could explain the high resistance to chemotherapy. Novel therapies targeting the tumor microenvironment with an aim to decrease this resistance, improve immune tolerance and increase the efficacy of the current treatment have shown some promising preliminary results in preclinical and clinical trials. We review the current advances in the field of immunotherapy and their effectiveness as a potential treatment strategy in the pancreatic cancer.

Keywords

adoptive cellular immunotherapy cancer vaccines immunotherapy pancreatic cancer

Introduction

Pancreatic cancer is the twelfth most common cancer and is the seventh most common cause of cancer related deaths worldwide with 338,000 new cases and a 5-year prevalence of 4.1 per 100,000 [Ferlay et al. 2015]. In the United States, it is the third leading cause of cancer-related deaths with an estimated incidence of 48,960 cases, an estimated mortality of 40,560 in 2015 and 5-year survival rate of 7.2% [Howlader et al. 2015]. For many years, gemcitabine had been the primary first-line treatment for pancreatic ductal adenocarcinoma (PDAC). The advent of polychemotherapy approaches for locally advanced and metastatic disease using FOLFIRINOX (folinic acid, 5-fluorouracil, irinotecan, oxaliplatin) [Conroy et al. 2011] was able to attain a median survival (MS) of 11.1 months. Von Hoff and colleagues conducted a phase III MPACT trial to study efficacy and safety of nab-paclitaxel and gemcitabine combination versus monotherapy in pancreatic adenocarcinoma. The median overall survival (OS) was 8.5 months in the nab-paclitaxel–gemcitabine group as compared with 6.7 months in the gemcitabine group. The survival rate at 1 year was 35% and 22% and progression-free survival (PFS) was 5.5 and 3.7 months in combination versus monotherapy groups, respectively [Von Hoff et al. 2013]. More recently, Goldstein and colleagues updated results based on long-term follow up and found OS to be 8.7 versus 6.6 months in combination and monotherapy group and found long-term survivors (>3 years) in the combination group (4%) [Goldstein et al. 2015]. There have been many attempts to create other polychemotherapy regimens by combining additional agents with gemcitabine, without significant benefit noted. A randomized phase III trial of pemetrexed in combination with gemcitabine given to 565 locally advanced or metastatic pancreatic cancer demonstrated no significant survival benefits with a MS of 6.5 months and progression-free interval (PFI) of 3.6 months as compared with single-agent gemcitabine [Oettle et al. 2005]. Despite the advances in the treatment for PDAC, patients eventually experience disease progression and cures are rare.

The effect of gemcitabine on the immune system in advanced pancreatic cancer patients has been studied and was not found to be immunosuppressive. Regardless of decreased memory T cell, gemcitabine promotes naïve T-cell activation and may enhance responses to antitumor vaccines and immunotherapy [Plate et al. 2005]. Further effect of gemcitabine proceeded by pemetrexed, an antifolate drug, on innate and adaptive immunity was studied in a phase II study of 15 patients with advanced pancreatic cancer. The study demonstrated MS of 9.5 months and PFI of 4.75 months. Pemetrexed decreased the levels of natural killer (NK) cytolytic units when combined with gemcitabine and initially increased levels of interferon gamma (IFNγ) in NK cells which returned to baseline after the addition of gemcitabine. The combination led to decreased OX40+ activated T cells, helper T cells and memory T cells but levels were increased in subjects with high expression of B7-H3 in tumor tissue. Innate NK cell immunity and FoxP3+ T cells and CD8+ T cells correlated positively with survival and seemed beneficial in patients with pancreatic tumor. Thus, innate and adaptive immunity are important defenses against pancreatic tumors [Davis et al. 2012]. As such, there is a need to further explore novel therapies to improve morbidity and mortality related to PDAC.

Current progress in immunology-based therapy targets the highly heterogeneous pancreatic tumor immunosuppressive microenvironment. Macrophages, myeloid derived suppressor cells and regulatory T cells (Tregs) are the three major leukocyte subtypes found in early pancreatic intraepithelial (PanIN) stage that are involved in immunosuppressive antitumor activity and more advanced PDAC stage is associated with increased number of Tregs and inactivated effector T cells (Teffs) [Clark et al. 2007]. Various animal and human models have suggested that these immunosuppressive cells also increase in the peripheral blood, stroma and accumulate in PDAC tissue hindering the normal effector T-cell proliferation and response [Liyanage et al. 2002; Zhao et al. 2009; Amedei et al. 2013]. An animal model demonstrated that manipulation of these targets may inhibit or restrict tumor growth [Tan et al. 2009]. These observations advocate for the role of immunosuppression in PDAC. In this article, we review recent advances in immunotherapeutics for pancreatic cancer treatment. See Figure 1 for a diagrammatic depiction of mechanisms.

Figure 1.

Illustrative image describing immunotherapy mechanisms employed in defense against pancreatic cancer.

Immune checkpoint inhibitors

Tumor peptide antigen presentation by major histocompatibility complex (MHC) to the T-cell receptor (TCR) is helpful in eliciting the antitumor immune response. However, this response is modulated by several regulatory molecules expressed on T cells termed ‘immune checkpoints’. These costimulatory or coinhibitory molecules synergize with TCR signaling to promote or inhibit the immune activity under physiologic and pathologic conditions. Co-regulatory molecules serve as immunotherapeutic targets to modify MHC–TCR signaling to optimize the immune response against cancer cells [Pardoll, 2012; Chen and Flies, 2013].

Anti-CTLA-4

CTLA-4 (CD152) is one of the first immune checkpoint receptors to be targeted clinically [Brunet et al. 1987]. It is involved in regulating early stages of T-cell activation. It belongs to the family of CD28 receptors and has an inducible expression on the surface of CD8+ and CD4+ T cells. CTLA-4 binds to ligands B7-1 (CD80) and B7-2 (CD86) on antigen-presenting cells (APCs), and due to its higher avidity, displace the costimulatory receptor CD28. Thus, by blocking the stimulatory effect of CD28 ligand, it attenuates the immune response by inhibiting activation of Teffs, decreasing interleukin (IL)-2 production and activating Tregs to exert suppression [Blank and Enk, 2015]. Several other extrinsic and intrinsic cell mechanisms are also believed to play a role in CTLA-4 function. These include reverse signaling via B7 that can lead to increased production of indoleamine-2,3-dioxygenase (IDO) decreasing T-lymphocyte activation and proliferation and presence of associated phosphatases SHP-2 and PP2A impairing TCR signaling by dephosphorylation leading to overriding of the TCR-induced stop signal required for stable conjugate formation between T cells and APCs, ultimately leading to reduced cytokine production and proliferation [Schneider et al. 2006; Rudd, 2008]. Collectively, all of these factors are believed to limit the body’s immune response to antigens including self-antigens.

The tumor microenvironment in pancreatic cancer has an increased Tregs/Teffs [Ino et al. 2013] and increased expression of negative costimulatory molecules [Chen and Flies, 2013]. Preclinical murine models demonstrated improved tumor control and its shrinkage after CTLA-4 blockade [Leach et al. 1996; Van Elsas et al. 1999; Peggs et al. 2009] which led to the clinical development of the anti-CTLA-4 agent, ipilimumab [Hodi et al. 2010]. Improvement of OS by 4 months (10 versus 6.4 months) led to US Food and Drug Administration (FDA) approval of ipilimumab in metastatic melanoma.

Ipilimumab in pancreatic cancer

Ipilimumab is a fully humanized IgG1 monoclonal antibody (mAb) that antagonizes CTLA-4. A phase II trial [Royal et al. 2010] evaluated the efficacy of ipilimumab for advanced pancreatic cancer in 27 subjects with good performance status. An intravenous dose of 3.0 mg/kg every 3 weeks, four doses per course for a maximum of two courses were given. There were no responders by Response Evaluation Criteria in Solid Tumors (RECIST) criteria but one subject experienced a delayed tumor regression after an initial assessment of progressive disease. Given that RECIST or World Health Organization (WHO) criteria may not provide complete assessment of antitumor response with immunotherapeutics or other such novel agents, a systematic criterion classified as immune-related response criteria was defined to capture and to better interpret any additional atypical responses observed with immune therapy [Wolchok et al. 2009].

A phase Ib trial [ClinicalTrials.gov identifier: NCT00836407] was performed to determine clinical safety, survival rates and T-cell responses to ipilimumab 10 mg/kg alone (arm 1) and in combination with granulocyte macrophage colony-stimulating factor (GM-CSF) vaccine GVAX (arm 2) in patients with previously treated PDAC. Two patients in arm 1 and three patients in arm 2 demonstrated stable disease, and seven patients in arm 2 noted a decline in CA19-9. The combination arm demonstrated an improved median OS (3.6 versus 5.7 months) and 1-year OS (7% versus 27%) [Le et al. 2013]. Additional investigation into optimal use, clinical safety and efficacy of ipilimumab alone or in combination has led to several ongoing clinical trials as documented in Tables 1 –5.

Table 1.

Strategy-immune checkpoint inhibitor monotherapy.

Intervention	Strategy	Date (last updated)	Status	Patient population	Phase	Study design	ClinicalTrials.gov identifier/ Reference
Ipilimumab	CTLA-4 inhibitor	23 September 2015	Completed	Locally advanced or metastatic pancreatic adenocarcinoma	II	Single arm Ipilimumab	NCT00112580
Pembrolizumab	PD-1 inhibitor	10 December 2015	Recruiting	Advanced cancer including pancreatic	Ib/II	Single-agent dose-escalation study	NCT02331251
Pembrolizumab (MK3475)	PD-1 inhibitor	23 November 2015	Recruiting	Resectable borderline resectable pancreatic cancer	I/II	Arm 1- pembrolizumab + Neoadjuvant chemoradiation (CRT) Arm 2- Neoadjuvant CRT capecitabine alone	NCT02305186
Pembrolizumab	PD-1 inhibitor	2 September 2015	Recruiting	Advanced solid tumors	I	Single-arm Pembrolizumab	NCT02054806
Pembrolizumab	PD-1 inhibitor	16 April 2015	Recruiting	Metastatic cancers including pancreatic adenocarcinoma	I	Pembrolizumab with hypofractionated radiotherapy	NCT02303990
MPDL3280A	PD-L1 inhibitor	1 December 2015	Recruiting	Locally advanced or metastatic solid tumors	I	Single-arm i.v. infusion of MPDL3280A	NCT01375842 / [Herbst et al. 2014]
MPDL3280A	PD-L1 inhibitor	11 November 2015	Recruiting	Solid tumors	II	Single-arm study of MPDL3289A	NCT02458638
MEDI4736	PD-L1 inhibitor	11 November 2015	Recruiting	Advanced solid tumors	I/II	Single-arm dose-expansion study	NCT01693562
Nivolumab + Lirilumab (anti-KIR2DL1/2L3 antibody)	PD-1 Inhibitor, Anti-Killer cell immunoglobulin like receptors	3 July 2015	Recruiting	Advanced solid tumors	I	Dose-escalation study	NCT01714739
Nivolumab (BMS-936558) + BMS-986016 (anti-LAG-3)	PD-1 inhibitor, Anti lymphocyte activating gene-3	8 December 2015	Recruiting	Solid tumors	I	Part A-Dose escalation: BMS-986016 Part B-Dose escalation: BMS-986016 + BMS-936558 Part C-Cohort expansion: BMS-986016 + BMS-936558	NCT01968109

Table 2.

Strategy-dual checkpoint inhibitor combinations.

Intervention	Strategy	Date (last updated)	Status	Patient population	Phase	Study design	ClinicalTrials.gov identifier
Nivolumab + Ipilimumab	PD-1 inhibitor, CTLA-4 inhibitor	8 December 2015	Recruiting	Advanced metastatic pancreatic tumor	I/II	Arm N- Nivolumab monotherapy. Arm N-I – Nivolumab + Ipilimumab combination therapy	NCT01928394
Tremelimumab + MEDI4736	CTLA-4 inhibitor, PD-L1 inhibitor	5 December 2015	Recruiting	Unresectable pancreatic cancer	I	Arm A- MEDI4736 + SBRT Arm B- Tremelimumab + SBRT Arm C-MEDI4736 + Tremelimumab + SBRT	NCT02311361
Tremelimumab + MEDI4736	CTLA-4 inhibitor, PD-L1 inhibitor	7 December 2015	Recruiting	Advanced solid tumors (Urothelial bladder/ Triple negative Breast cancer/ PDAC)	II	Arm 1- Tremelimumab monotherapy Arm 2- MEDI4736 monotherapy Arm 3- Tremelimumab + MEDI4736 combination	NCT02527434
Tremelimumab + MEDI4736 + Mogamulizumab (anti- CCR-5)	CTLA-4 inhibitor, PD-L1 inhibitor, Anti chemokine receptor 5	4 December 2015	Recruiting	Advanced solid tumors	I	Arm1- Mogamulizumab + MEDI4736 Arm2- Mogamulizumab + Tremelimumab	NCT02301130

Table 3.

Strategy-immune checkpoint inhibitors and vaccine combination therapy.

Intervention	Strategy	Date (last updated)	Status	Patient population	Phase	Study design	ClinicalTrials.gov identifier
Ipilimumab + GVAX	CTLA-4 inhibitor, Whole cell vaccine	21 October 2015	Recruiting	Metastatic Pancreatic cancer	II	Arm1- Ipilimumab + GVAX Arm2- FOLFIRINOX. Endpoint – Efficacy study	NCT01896869
Nivolumab + GVAX + Cyclophosphamide	PD-1 inhibitor, Whole cell vaccine, Cytotoxic drugs	1 December 2015	Open but not recruiting	Surgically resectable pancreatic cancer	I/II	Arm A- CY/ GVAX Arm B- CY/ GVAX + Nivolumab	NCT02451982
Nivolumab+ GVAX+ CRS-207	PD-1 inhibitor, Whole cell vaccine, Listeria vaccine	23 September 2015	Recruiting	Metastatic pancreatic cancer	II	Arm 1 – CY/ GVAX/ CRS-207/ Nivolumab Arm 2- CY/ GVAX/ CRS-207	NCT02243371

Table 4.

Strategy-immune checkpoint inhibitors with chemotherapy.

Intervention	Strategy	Date (last updated)	Status	Patient population	Phase	Study design	ClinicalTrials.gov identifier
Ipilimumab + Gemcitabine	CTLA-4 inhibitor, Cytotoxic drugs	10 November 2015	Recruiting	Locally Advanced, unresectable or metastatic pancreatic cancer	Ib	Safety study with induction and maintenance doses of Ipilimumab and Gemcitabine	NCT01473940
Nivolumab + Nab-Paclitaxel + Gemcitabine	PD-1 inhibitor, Cytotoxic drug	20 October 2015	Recruiting	Pancreatic cancer	I	Arm A- Nivolumab + nab-paclitaxel Arm B- Nivolumab + nab-paclitaxel + Gemcitabine	NCT02309177
Pembrolizumab (MK-3475) + mFOLOFOX6	PD-1 inhibitor	11 December 2015	Recruiting	Advanced GI cancer	I/II	Arm- MK-3475 in combination with mFOLFOX6 in a 14 day cycle	NCT02268825
Pidilizumab (CT-011) + Gemcitabine	PD-1 inhibitor	15 July 2015	Not recruiting	Resected pancreatic cancer	II	Single arm with CT-011 + Gemcitabine combination therapy	NCT01313416
MPDL3280A + Bevacizumab +/- FOLFOX, Carboplatin, Nab-paclitaxel, Pemetrexed	PD-L1 inhibitor, anti-VEGF antibody, Cytotoxic drugs	1 December 2015	Recruiting	Locally advanced or metastatic solid tumors	I	Multiple arms accessing safety and maximum tolerated doses	NCT01633970

Table 5.

Strategy-immune checkpoint inhibitors and tyrosine kinase inhibitors combination therapy.

Intervention	Strategy	Date (last updated)	Status	Patient population	Phase	Study design	ClinicalTrials.gov identifier
Ipilimumab + Imatinib mesylate	CTLA-4 inhibitor, Tyrosine kinase inhibitor	30 November 2015	Recruiting	Advanced cancer	I	Combination therapy of Ipilimumab with Dose Escalation of Imatinib mesylate	NCT01738139
Pembrolizumab + ACP-196	PD-1 inhibitor, Bruton tyrosine kinase inhibitor	30 November 2015	Active but not recruiting	Advanced or metastatic pancreatic cancer	II	Arm1- ACP-196 alone Arm2- ACP-196 + Pembrolizumab	NCT02362048
Pembrolizumab + PLX3397	PD-1 inhibitor, Tyrosine kinase inhibitor	9 October 2015	Recruiting	Advanced solid tumors including pancreatic cancer	I	Arm- PLX3397 + pembrolizumab combination therapy	NCT02452424
MEDI4736 + Ibrutinib mesylate	PD-L1 inhibitor, Tyrosine kinase inhibitor	4 September 2015	Recruiting	Refractory or relapsed solid tumors	Ib/II	Phase Ib- MEDI4736+ Ibrutinib to determine the recommended phase II dose level Phase II- MEDI4736+ Ibrutinib at recommended phase II dose level	NCT02403271

Tremelimumab in pancreatic cancer

Tremelimumab is a human IgG2 mAb that blocks CTLA-4 [Ribas et al. 2007]. Phase II/III clinical trials in patients with advanced melanoma treated at a dosage of 15 mg/kg every 3 months failed to yield any significant survival benefit [Kirkwood et al. 2010; Ribas et al. 2013]. A phase II single-arm study in patients with malignant mesothelioma yielded a PFS of 6.2 months and a median OS of 10.7 months [Calabrò et al. 2013]. Clinical trials for tremelimumab in pancreatic cancer are underway.

Anti-PD1 and anti-PD-L1

Programmed cell death protein-1 (PD-1)/CD279 is a coinhibitory molecule of the CD28 family. It is expressed predominantly by activated CD4+ and CD8+ T cells, Tregs [Francisco et al. 2010] and is also found on activated B cells [Pedoeem et al. 2014], NK cells, monocytes and certain dendritic cells (DCs). Its binds to the PD-L1 (B7-H1) and PD-L2 (B7-DC) ligands of family B7. PD-L1 is constitutively expressed on several solid tumor cells and tumor-infiltrating lymphocytes (TILs) [Dong et al. 2002] while PD-L2 is expressed on DCs, macrophages, B-cell subsets [Pedoeem et al. 2014] and is upregulated in lymphomas and hematologic malignancies [Rosenwald et al. 2003]. PD-1 engagement with its ligands controls peripheral T-cell tolerance. PD-L1 interaction with PD-1 and B7-1 (CD80) results in negative signals leading to decreased T-cell proliferation, cytokine release and cytolytic activity of PD-1+ T cells. Chronic antigen exposure such as cancer and viral infection, can lead to persistent PD-1 expression inducing a state of exhaustion among antigen-specific T cells and helping the tumor cells escape from host immune attack. Blockade of these targets reverses the tumor protective effects and may provide an effective approach for tumor immunotherapy [Iwai et al. 2002; Okudaira et al. 2009]. Increased expression [Razzaque et al. 2013] of PD-L1 and PD-L2 significantly correlates with worse prognosis, higher grade and is inversely correlated with TILs in pancreatic cancer [Nomi et al. 2007; Geng et al. 2008]. Furthermore, its role in pancreatic adenocarcinoma is defined, given that knocking down of B7-H1 (PD-L1) inhibits PDAC proliferation [Song et al. 2014].

Nivolumab and pembrolizumab, examples of anti-PD1 therapeutic agents, have demonstrated positive results with in various studies involving malignant melanoma [Mahoney et al. 2015], renal cell carcinoma [Weinstock and Mcdermott, 2015] along with preliminary efficacy in a wide array of other tumors. Pembrolizumab has been approved by US FDA for malignant melanoma previously treated with ipilimumab [Robert et al. 2014]. The first PD-L1 antibody to show an objective response in a phase I study including patients with solid tumors was BMS-956559 [Brahmer et al. 2012], but no responses in patients with pancreatic and colorectal cancer were observed. Various PD-1 and PD-L1 inhibitors are presently in clinical trials for pancreatic malignancies.

A broad array of clinical trials in pancreatic cancer alone or advanced solid tumors including pancreatic cancer have been completed or ongoing using immune checkpoint monotherapy (Table 1), dual checkpoint combination therapy (Table 2), immune checkpoint inhibitor and vaccine combinations (Table 3), immune checkpoint inhibitors with cytotoxic chemotherapy (Table 4), immune checkpoint inhibitors with kinase inhibitors (Table 5) and immune checkpoint inhibitor combinations with other agents (Table 6).

Table 6.

Strategy-other therapies in combination with immune checkpoint inhibitors.

Intervention	Strategy	Date	Status	Patient population	Phase	Study design	ClinicalTrials.gov identifier
Nivolumab + FPA008 (CSF1R inhibitor)	PD-1 inhibitor, Colony stimulating factor-1 receptor inhibitor	2 December 2015	Recruiting	Advanced pancreatic cancer	I	Arm 1- Phase 1a monotherapy Arm 2- Phase1a combination therapy to determine safety data Arm 3- Phase 1b combination therapy to determine clinical activity and safety profile	NCT02526017
Nivolumab + Ulocuplumab (anti- CXCR4)	PD-1 inhibitor, Anti alpha chemokine receptor 4	8 December 2015	Recruiting	Metastatic solid tumors	I/II	Combination arm in pancreatic cancer	NCT02472977
Pembrolizumab + AM0010	PD-1 inhibitor, PEGylated recombinant human IL-10	24 March 2015	Recruiting	Advanced solid tumor including pancreatic cancer	I	Combination therapy	NCT02009449
MPDL3280A + RO6895882(IL-2v with anti-CEA)	PD-L1 inhibitor, Variant of Interleukin-2 against Carcinoembryonic antigen	1 December 2015	Recruiting	Advanced solid tumors	I	Part 1- Dose Escalation RO6895882(IL-2v with anti-CEA) + MPDL3280A Part 2- Dose expansion	NCT02350673
Pembrolizumab+ Modified vaccinia virus Ankara virus	PD-1 inhibitor, Oncolytic virus	18 December 2015	Not recruiting	Solid tumors with failed prior therapy including PDAC	I	Combination therapy	NCT02432963

Therapeutic vaccine immunotherapy

The principle of using vaccine immunotherapy in cancer treatment is similar to passive immunity against an infectious process. These are biologically designed to mount an immune response against a tumor by administering tumor-specific antigens. They include whole-cell vaccines, DC vaccines, DNA vaccines and peptide vaccines.

Whole-cell vaccines

Whole-cell recombinant vaccines utilize the tumor cells which have a wide range of antigens and express epitopes for CD8+ and CD4+ T cells. Allogeneic cell lines may be used instead of autologous cell lines in pancreatic cancer when it is difficult to harvest these tumor cells due to limited number of surgical candidates, long culture times and risk of contamination [Koido et al. 2005].

Algenpantucel-L

Algenpantucel-L is a hyperacute vaccine that has allogenic irradiated pancreatic cancer cells expressing the enzyme alpha-1,3-galactosyl transferase (αGT) which is required for the synthesis of alpha-galactosyl (αGal) epitopes (Table 7). Humans lack the presence of αGal epitopes but have anti-αGal antibodies [Galili et al. 1985] and these are continuously produced in response to enterobacteria with no immune tolerance to it [Galili et al. 1988]. The hyperacute technology exploits this naturally acquired immunity against αGal labeled tumor cells leading to enhanced antitumor response [Mandell et al. 2009]. Effective tumor responses in animal models [Gorelik et al. 1995; Rossi et al. 2005] led to subsequent human clinical trials. Hardacre and colleagues [Hardacre et al. 2012], presented results of an open-label, single-arm phase II study of algenpantucel-L with gemcitabine and 5-fluorouracil for 70 resected pancreatic cancer patients. One-year disease-free survival (DFS) of 63% and OS of 86% was achieved as compared with historical controls (~45% and 65%). Current ongoing phase III clinical trials are the Immunotherapy for Pancreatic Resectable Cancer Survival Study (IMPRESS) and the Pancreatic Immunotherapy with algenpantucel-L for Locally Advanced non-Resectable (PILLAR) trial.

Table 7.

Algenpantucel-L in clinical trials.

Intervention	Strategy	Date (last updated)	Status	Patient population	Phase	Study design	ClinicalTrials.gov identifier (acronym)
Algenpantucel-L + Gemcitabine +/– 5-FU	Hyperacute (HAPa) vaccine, Cytotoxic drugs	5 August 2015	Ongoing but not recruiting	Surgically resected pancreatic cancer	III	Randomized double arm. Arm 1 – HAPa Vaccine + Cytotoxic drugs. Arm 2 – Cytotoxic drugs	NCT01072981 (IMPRESS)
Algenpantucel-L + FOLFIRINOX + Gemcitabine + 5-FU chemoradiation + Capecitabine + Nab-paclitaxel	HAPa, Cytotoxic drugs, Chemoradiation	28 October 2015	Recruiting	Borderline resectable or locally advanced unresectable pancreatic cancer	III	Arm 1A – FLOFIRINOX + HAPa Arm 1B – Gemcitabine/Nab-Paclitaxel + HAPa Vaccine Arm 2A – FOLFIRINOX alone Arm 2B – Gemcitabine/Nab-paclitaxel	NCT01836432 (PILLAR)
Algenpantucel-L + mFOLFIRINOX + Gemcitabine + SBRT	HAPa, Cytotoxic drugs, Radiation	23 September 2015	Recruiting	Borderline resectable pancreatic cancer	II	Single-arm combination therapy	NCT02405585

GM-CSF vaccine

GVAX is an allogenic irradiated whole-cell tumor vaccine transfected with GM-CSF gene and delivering tumor-associated antigens (TAAs) at the same time (Table 8). GM-CSF is a potent cytokine released by macrophages, T cells, mast cells, NK cells that function to stimulate stem cells to produce granulocytes and monocytes, promoting cytolytic activity against tumor cells [Thomas et al. 2004] and also causing influx of bone-marrow derived DCs that process and present TAAs delivered by the vaccine [Huang et al. 1994]. A phase I [Jaffee et al. 2001] clinical trial of GVAX in resectable pancreatic cancer given before and after chemoradiation therapy, assessed 14 patients and showed a mean DFS of 13 months. Three patients who demonstrated delayed-type hypersensitivity (DTH) were disease free from 25 to 30 months. A dose of 50 × 10⁷ vaccine cells was identified as safe with limited side effects and associated with induction of antitumor immune response. These favorable results resulted in phase II trials of GVAX in combination with cyclophosphamide (CY) [Laheru et al. 2008] in metastatic pancreatic cancer patients who failed gemcitabine therapy. Despite no significant statistical difference of OS and 1-year survival between two cohorts, this study suggested enhanced antitumor activity in combination cohort where CY is given prior to immunotherapy based on a higher rate of induction of mesothelin-specific T-cell responses that could be associated with longer PFS and OS. Another phase II study of 60 patients with resected PDAC received GVAX integrated with boosters and 5-FU-based chemoradiation demonstrated a median DFS of 17.3 months with MS of 24.8 months. It also demonstrated a correlation between induction of mesothelin-specific T-cell responses with improved OS proposing a prognostic role [Lutz et al. 2011]. Additional markers that may be associated with prolonged survival in patients treated with GVAX and/or ipilimumab are induction of thyroglobulin antibodies due to thyroid autoimmunity [De Remigis et al. 2015].

Table 8.

Clinical trials that used combination of whole cell vaccine GVAX and chemotherapy.

Intervention	Strategy	Date (last updated)	Status	Patient population	Phase	Results	ClinicalTrials.gov identifier
GVAX + Adjuvant standard of care chemoradiation.	Whole-cell vaccine, Cytotoxic drugs	17 July 2015	Completed	60 patients with resected stage I or II pancreatic adenocarcinoma	II	Overall survival – 24.8 months. Disease-free survival – 17.3 months	NCT00084383
GVAX + Cyclophosphamide + Cetuximab	Whole-cell vaccine, Cytotoxic drugs	18 June 2015	Completed	60 patients with metastatic or locally advanced pancreatic cancer	II	Total serious adverse events – 12/60 (20%)	NCT00305760
GVAX + Cyclophosphamide.	Whole-cell vaccine, Cytotoxic drugs	17 November 2105	Ongoing not recruiting	Stage I or II pancreatic cancer	Pilot	Combination versus GVAX monotherapy.	NCT00727441
GVAX + Cyclophosphamide + SBRT + FOLFIRINOX	Whole-cell vaccine, Cytotoxic drugs, Radiation	27 July 2015	Ongoing not recruiting	Pancreatic cancer	Pilot	Arm 1 - SBRT + FOLFIRINOX. Arm 2 - Combination therapy	NCT01595321

Peptide vaccines

Peptide-based vaccines constitute tumor antigenic fragments (epitopes) which elicit a tumor-specific response by stimulating T cells. These vaccines are safe, simple and economically beneficial. However, their minimal immunogenic potential, impaired antigen presentation [Yanagimoto et al. 2005] and defective immune response in patients with advanced cancer have limited their usefulness [Gaudernack, 2006].

Ras vaccine

K-Ras is mutated in almost 90% of pancreatic cancers [Hruban et al. 1993] and is presented as a foreign antigen by MHC class I and II to CD4+ and CD8+ T cells [Abrams et al. 1997; Gjertsen et al. 1997]. Therefore, K-Ras mutated gene product can serve as a focus for therapeutic peptide vaccine inducing anti-Ras immunity in patients. Initial studies testing Ras vaccine in humans [Gjertsen et al. 1995] demonstrated the safety and induced transient T-cell tumor response against mutations in tumor cell, albeit with no clinical responses. Failure to manifest clinically significant immunity was attributed to the inability to elicit the imperative immune response to these antigens. Eventually in an attempt to enhance the immune response, a phase I/II trial combining synthetic mutant Ras peptides with GM-CSF as an adjuvant was given to 48 patients (10 surgically resected and 38 with advance disease) of pancreatic adenocarcinoma. Peptide-specific immunity was induced in 58% of patients and the responders showed an improved survival over nonresponders (MS 148 days versus 61 days, respectively) and 9 of 10 resected PDAC patients showed stability with an OS of 25.6 months (range 10–39 months) and 11 of 34 unresectable patients had a stable disease lasting an average of 10.2 months (range 3–28 months) [Gjertsen et al. 2001].

Adjuvant use of Ras vaccine in a phase II clinical trial [Toubaji et al. 2008] in patients with surgically resected (five pancreatic and seven colorectal cancer patients) with no evidence of disease at enrollment demonstrated a positive immune response with an increase of IFNγ m-RNA copy numbers in 5/11 patients. Pancreatic cancer patients had a mean DFS of 35.2+ months and a mean OS of 44.4+ months providing evidence for adjuvant early use of mutant Ras peptide immunotherapy. Subsequently [Wedén et al. 2011], a study using a long synthetic mutant Ras peptide vaccine described a 10-year follow up of 23 patients from previous phase I/II clinical trials [Carbone et al. 2005; Toubaji et al. 2008] to assess their long-term immunological T-cell reactivity and survival. A total of 17 of 20 (85%) were immune responders with a MS of 28 months. The 5-year survival was 22% and 28% for all and immune responders respectively and a striking 10-year survival of 20% (4/20 evaluable) versus 0% in comparable nonvaccinated cohort, was observed. Recently, another study evaluated 24 patients with resected pancreatic cancer with K-Ras mutation that were vaccinated with peptide vaccine on day 7 of a 10-day course of GM-CSF. A total 25% of patients (9/24) were immune responders, three of them displayed nonspecific DTH and one patient had a K-Ras mutation-specific DTH. Median recurrence-free survival time was 8.6 months and median OS was 20.3 months [Abou-Alfa et al. 2011]. These studies demonstrate promising results and suggest larger controlled studies depicting the role of K-Ras vaccine as an adjuvant immunotherapy for PDAC are needed.

Another strategy applied to activate immune system against the mutated Ras peptide is by using nonpathogenic yeast as a vehicle to carry antigens and present them to DCs. DCs treated with yeast and cocultured with T cells had a lower expression of FoxP3 cells and increase in the ratio of CD4+CD25+ activated T cells to Tregs and production of Th1-related cytokines and IL-6 [Cereda et al. 2011]. GI-4000 is a product series of four tarmogens; each tarmogen is a different heat inactivated yeast Saccharomyces cerevisiae expressing combination of common Ras mutations found in human cancers. Clinical trials studying the K-Ras peptide vaccine alone or as combination therapy in pancreatic cancer are shown in Table 9.

Table 9.

Ras vaccine in clinical trials.

Intervention	Strategy	Date	Status	Patient population	Phase	Study design	ClinicalTrials.gov identifier
Ras vaccine, Detox-B adjuvant	Peptide cancer vaccine, Immunostimulant	27 April 2015	Completed	Pancreatic, Colon or Lung cancer	I	Adjuvant treatment for Stage I–IV resected pancreatic cancer	NCT00019006
Ras vaccine, P53 peptide vaccine, Aldesleukin, Sargramostim, Therapeutic autologous and TILs	Peptide cancer vaccine, DC vaccine, IL-2, Leukocyte growth factor	19 June 2013	Completed	Advanced solid tumors	II	Vaccine therapy alone or in combination with cellular immunotherapy	NCT00019084
Ras vaccine, Sargramostim, Aldesleukin, Detox PC	Peptide vaccine, Leukocyte growth factor, IL-2, Immunostimulant	19 July 2013	Completed	Metastatic solid tumors	II	Vaccine therapy alone or in combination	NCT00019331
Ras vaccine, QS21	Peptide vaccine, Natural saponin stimulant	10 July 2013	Completed	Advanced pancreatic or colorectal cancer	I	Safety study	NCT00006387
GI-4000, Gemcitabine	Globeimmune heat killed yeast cell transfected with mutated Ras peptide, Chemotherapy	2 April 2015	Completed	Nonmetastatic, post-resection PDAC	II	Efficacy of vaccine compared with placebo	NCT00300950

Telomerase peptide vaccine

Telomerase is a ribonucleoprotein enzyme found in almost 90% cancer cells [Vasef et al. 1999] including pancreatic cancer cells [Suehara et al. 1997]. The induction of human telomerase reverse transcriptase (hTERT), a telomerase catalytic subunit parallels the increased expression of the enzyme [Counter et al. 1998]. Acknowledging its role in tumorigenesis, telomerase tumor antigen served as a target for the development of peptide-based vaccine, GV1001. It is a 16-aa hTERT peptide that binds to MHC activating hTERT-specific T-cell responses [Su et al. 2005]. GV1001 was studied in a phase I/II trial by Bernhardt and colleagues in 48 patients with unresectable pancreatic cancer. The GV1001 vaccine was given in three dose levels (100, 300, 1000 nmol) along with GM-CSF for a period of 10 weeks with monthly boosters. Immune response (positive DTH test or the presence of GV1001-specific T cells in peripheral blood after vaccination) was observed in 24 of 38 evaluable patients with the highest immune response (75%) in the intermediate dosage group. The MS for the low-, intermediate- and high-dose groups were 4.0, 8.6 and 5.1 months, respectively. As such, prolonged immunologic response correlated with prolonged survival in the study [Bernhardt et al. 2006].

The two phase III trials testing PrimoVax (GV1001 and GVAX) [Buanes et al. 2009] and TeloVac (GV1001) [Middleton et al. 2013] administered with subsequent and concurrent chemotherapy were unable to simulate similar affirmative results and showed no significant survival benefits in patients with metastatic pancreatic cancer. A currently ongoing clinical trial [ClinicalTrials.gov identifier: NCT01342224] is testing GV1001 with GM-CSF in patients with locally advanced pancreatic cancer given in combination with gemcitabine chemotherapy and tadalafil (PDE5 inhibitor) followed by concurrent radiation therapy.

Mucin-1 vaccine

Mucin 1 (MUC-1) is type I transmembrane protein constituting a large extracellular domain with extensive O-linked glycosylation and a small cytoplasmic domain in close contact with the nucleus and plays a role in normal cell signal transduction as well as oncogenic signaling to increase invasion, angiogenesis and metastasis [Behrens et al. 2010]. MUC-1 expression is increased in tumor cells including pancreatic tumors [Burdick et al. 1997; Qu et al. 2004] with a loss of polarity and hypoglycosylation. The overexpression and aberrant glycosylation makes MUC-1 highly immunogenic and, thus, a target for cancer immunotherapy [Besmer et al. 2011]. MUC-1 vaccine has been tested in different forms such as peptide vaccine, peptide transfected DC, DCs transfected with c-DNA22 tandem repeats units of MUC-1, as well as adoptive transfer immunotherapy. The phase I clinical trials by Goydos and colleagues [Goydos et al. 1996], Ramanathan and colleagues [Ramanathan et al. 2005] and most recently Yamamoto [Yamamoto et al. 2005] used peptide-based vaccines. Pecher and colleagues [Pecher et al. 2002] tested three different types of formulation of mucin vaccine namely: (1) MUC-1 peptide admixed with murine GM-CSF as an adjuvant; (2) MUC-1 peptide admixed with adjuvant SB-AS2; and (3) MUC-1 peptide-pulsed DCs. Only MUC-1 peptide loaded DC vaccine was able to elicit tumor rejection and response correlated with induction of MUC-1-specific T-cell response in wild and transgenic mice. This was further tested by Lepisto and colleagues [Lepisto et al. 2008].

These studies (Table 10) that define the effect of MUC-1 immunotherapy on pancreatic cancer reveal the safety of the vaccine and has led to increased survival in few vaccinated subjects. MUC-4, MUC-16 are the other potential targets that play a role in PDAC development and also lead to resistance of chemotherapeutic drugs [Bafna et al. 2009; Skrypek et al. 2013] Overcoming this resistance by combining chemotherapeutics with MUC based therapy may improve the clinical outcomes of patients with pancreatic cancer.

Table 10.

MUC-1 vaccine in clinical trials.

Intervention	Adjuvant	Strategy	Phase	Patient population	Year	Outcomes	Reference
MUC-1 105 amino acid	BCG	Peptide-based	I	Pancreatic adenocarcinoma	1996	Vaccine well tolerated with strong T-cell immune response but no clinical response	Goydos et al. [1996]
Autologous DC transfected with 22 tandem repeat units of MUC-1		Gene transfected DC Vaccine	I/II	Advanced pancreatic adenocarcinoma	2002	Demonstrated feasibility and safety of vaccine	Pecher et al. [2002]
MUC-1 100 aa peptide	SB-AS2	Peptide-based vaccine	I	Resected and locally advanced pancreatic cancer	2005	Vaccine specific DTH response in 3/10 patients	Ramanathan et al. [2005]
MUC-1 100 aa peptide	Extracellular tandem repeats and incomplete Freund	Peptide-based vaccine	I	Advanced pancreatic and biliary cancer	2005	PDAC patients with high frequency of IFNγ secreting CD8+ T cells	Yamamoto et al. [2005]
MUC-1 peptide loaded DC		DC vaccine	I/II	Resected pancreatic and biliary tumors	2008	4/12 alive at 4 years without recurrence. Median survival is 26 months	Lepisto et al. [2008]

Anti-VEGFR vaccine

Vascular endothelial growth factor receptor 2 (VEGFR2) is upregulated and essential in tumor angiogenesis. A phase I trial combining VEGFR2-169 with gemcitabine was conducted in advanced pancreatic cancer patients. A total of 15/18 patients developed immunological response (cytotoxic T lymphocyte and Treg response). A total of 15/18 (83%) developed immunological reactions at the injection sites. Specific cytotoxic T lymphocytes (CTLs) reacting to the VEGFR2-169 peptide were induced in 11 (61%) of the 18 patients. The disease control rate was 67%, and median OS time was 8.7 months [Miyazawa et al. 2010]. A phase I study with pancreatic cancer patients testing gemcitabine with antiangiogenic peptide vaccine (VEGFR2-169) NCT00622622 and a phase I/II study [ClinicalTrials.gov identifier: NCT00655785] with VEGFR2-169 and VEGF1-1084 has been completed.

Survivin-based vaccine

Survivin is one of the cancer-specific proteins overexpressed in tumor cells and human fetal tissues, but hardly detectable in normal human tissues. It promotes angiogenesis, inhibits apoptosis and enhances proliferation [Ryan et al. 2009]. Expression of survivin is increased on pancreatic adenocarcinoma tissue as well as in serum [Dong et al. 2015] and is associated with poor prognosis and resistance to chemotherapy and radiation [Kami et al. 2004]. A 77-year-old patient suffering from pancreatic cancer with liver metastasis refractory to gemcitabine therapy received a survivin-based vaccine (consisting of 100 μg of a modified HLA-A2 restricted surviving (96–104) epitope in Montanide(R)) and achieved a complete remission of liver metastasis at 8 months but with recurrence of disease after being weaned from the vaccine [Wobser et al. 2006]. This effect was further tested in a study [Ishizaki et al. 2011] on the Pan02 mouse pancreatic adenocarcinoma cell line that combined gemcitabine and modified vaccinia Ankara (MVA) expressing murine survivin. This study resulted in significant tumor regression and prolonged survival. A total of 50% of the mice in the MVA-survivin + gemcitabine group were alive at 50 days, whereas all other animals in MVA-survivin and MVA-control + gemcitabine groups expired by 38 days post-tumor induction. These results observed with combined immunotherapy were primarily dependent on generation of CD8+ T cells. Gemcitabine and survivin act in synergy creating an antitumor environment by generating effector cells against self-tumor antigen and apoptosis of tumor cells that helps to reduce tolerance against survivin and prolong the survival in combination therapy. The data from the study suggests combination therapy may find its potential for clinical benefit in pancreatic cancer.

Antigastrin vaccine

Gastrin-17 is a growth factor for pancreatic and other gastrointestinal malignancies. Antigastrin-17 vaccine (G17DT) has a gastrin immunogen with diphtheria toxoid (DT) carrier protein. The first phase II study testing G17DT recruited 30 patients with advanced pancreatic cancer, out of which 20 patients produced an antibody response. The MS for immune responders was 217 days and 121 days for nonresponders (p = 0.0023) [Brett et al. 2002]. A randomized clinical trial of 154 patients with advanced pancreatic cancer was well tolerated (2/79 (2.5%) G17DT patients versus 0/74 (0%) placebo patients developed sterile abscess at the injection site). The MS in the G17DT group was 138 days versus 78 days for the control group [Gilliam et al. 2004]. Subsequently, a randomized, multicenter, double-blind, placebo-controlled study of 154 patients with advanced pancreatic cancer gave MS of 151 days as compared with 82 days with placebo [Gilliam et al. 2012]. Another multicenter study evaluated the role of G17DT + gemcitabine versus placebo + gemcitabine in 394 patients with locally advanced, recurrent or metastatic adenocarcinoma of pancreas. The MS was 5.8 months in G17DT + gemcitabine group which was lower than the placebo + gemcitabine (6.6 months) and same PFS of 3.9 months [Shapiro et al. 2005]. A prospective randomized phase III study testing G17DT for the treatment of advanced pancreatic cancer [ClinicalTrials.gov identifier: NCT02118077] was recently completed and results are awaited.

Heat shock protein–peptide complex

Heat shock proteins act as peptide carriers for stabilization and delivery. Overexpression of HSP90 is observed in pancreatic carcinoma and HSP90 β is reported to inhibit apoptosis [Ogata et al. 2000]. A phase I pilot study of autologous heat shock protein vaccine HSPPC-96 in patients with resected pancreatic adenocarcinoma to determine tolerance and immune as well as clinical responses. A total of 3/10 patients were alive at 2.6, 2.7 and 5 years follow up [Maki et al. 2007]. A phase I clinical trial of immunotherapy with autologous tumor-derived gp96 heat shock protein-peptide complex in patients with resected pancreatic adenocarcinoma [ClinicalTrials.gov identifier: NCT00003025] has been completed and results are awaited.

Dendritic cell vaccine

DCs are the most potent APCs which function to process and present antigens with MHC to activate cytolytic and regulatory T-cell response [Koski et al. 2008]. These characteristics are being employed in development of DC vaccines to mount an antitumor response (Table 11). DC vaccines are designed to present TAAs like peptides, mRNA or cDNA or by fused autologous DC with tumor cells and means by which they are presented including: (a) nontargeted vaccine which consists of tumor-specific peptides or nucleic acids captured in vivo; (b) vaccines with ex vivo generated DCs loaded with antigens; (c) vaccines with tumor antigens coupled to anti-DC-antibodies to mount in vivo response [Palucka and Banchereau, 2013]. Intratumoral response with tumor RNA-pulsed DC was studied in C57BL/6 murine pancreatic tumor model inducing a specific T lymphocyte antitumor effect [Schmidt et al. 2003].

Table 11.

Studies using dendritic cell (DC) vaccines.

Intervention	Strategy	Patient population	Phase	Year	Results	Reference
CEA loaded DC	mRNA infested DC vaccine	3 patients with resected pancreatic adenocarcinoma following neoadjuvant chemoradiotherapy.	Pilot	2002	Well tolerated, no evidence of adverse eventsAll patients were alive without evidence of disease at more than 30 months from the original diagnosis	Morse et al. [2002]
Mutant p53 and K-Ras loaded DC	Peptide based DC vaccine	39 patients with pancreatic, lung, colon, breast, ovarian, gastrointestinal and other cancers	I	2005	10/38 evaluable patients had detectable CTL response, 16 patients with positive IFNγ after vaccinationFive experienced stable disease. Median survival time of 393 versus 98 days for a positive versus negative CTL response	Carbone et al. [2005]
Adenovirus encoding Interleukin 12 (AFIL-12) gene transfected DC	Engineered DC vaccine	17 patients with metastatic pancreatic (3), colorectal (5), liver (9)	Pilot	2005	Stable disease in two patients and partial response in one. Progression in eight patientsMarked increase of CD8+ T lymphocytes in 3/11 tumor biopsies analyzed	Mazzolini et al. [2005]
MUC-1 pulsed DC	Peptide based DC vaccine	20 patients with unresectable or recurrent pancreatic cancer	Pilot	2008	Complete response in one patient with lung metastasis. 5 patients had stable disease. Median survival was 9.8 months	Kondo et al. [2008]

Further clinical trials to determine the effectiveness of CEA RNA-pulsed DC vaccine alone [ClinicalTrials.gov identifier: NCT00004604] and in combination with chemotherapy gemcitabine [ClinicalTrials.gov identifier: NCT00547144] and chemokine modulatory regimen (CKM) [ClinicalTrials.gov identifier: NCT02151448] are in process.

Recombinant bacterial vaccine

Bacterial agents such as Listeria monocytogenes (LM) and Salmonella enterica typhimurium act as delivery systems for TAAs because of their ability to survive intracellularly [Paterson et al. 2010]. Listeria is a Gram-negative intracellular facultative bacterium which is actively phagocytosed by macrophages or other APCs. Once inside the phagosome it secretes Listeriolysin O (LLO) and phospholipase C which helps it to modify the phagosomes to larger compartment called spacious Listeria-associated phagosomes (SLAPS) [Birmingham et al. 2008] promoting its replication and release from the phagolysosome into the cytoplasm. LM-based immunotherapy utilizes live attenuated LM bacteria to act as a vaccine vector and deliver the TAAs to the APCs and hence eliciting an antitumor immune response.

Preclinical studies have shown the antitumor effect of the vaccine. A study in FVB/N mouse fusing LLO with HER-2/neu antigen delivered by LM showed the increased immunogenicity of self-antigens and provided tumor regression [Singh et al. 2005]. CRS-207 is a live attenuated vaccine of LM bacterium has two gene deletions that encode for virulence and is modified to express mesothelin. A phase I clinical trial [Le et al. 2012] tested ANZ-100 (live attenuated LM strain) and CRS-207 in advanced mesothelin expressing cancers with liver metastasis. CRS-207 induced both LLO and mesothelin-specific T-cell responses and chemokine induction were present at all doses while ANZ-100 induction of chemokines was dose dependent. While the study was statistically unable to show survival benefit, it revealed that 37% of the population survived for more than 15 months with 3 PDAC long-term survivors with minimal adverse events. A more recently completed randomized phase II study assessed safety and survival of CRS-207 with GVAX and low-dose CY for 90 metastatic pancreatic cancer patients [Le et al. 2015]. GVAX/CY inhibits Tregs and had shown earlier preclinical synergy with the vaccine that helps to induce innate and adaptive immunity. OS was 9.7 months with an improvement of 5.6% (2.2 months) as compared with first-line therapy gemcitabine plus nab-paclitaxel. The longer OS was associated with enhanced mesothelin-specific CD8 T-cell response. An ongoing phase II clinical trial is testing the safety and efficacy of GVAX (with CY) and CRS-207 compared with CRS-207 alone in adults with previously treated metastatic pancreatic adenocarcinoma [ClinicalTrials.gov identifier: NCT02004262].

Adoptive T-cell immunotherapy

Adoptive T-cell immunotherapy (ACT) employs the technique of genetically modifying the T cells and infusing them into tumor tissue to increase the immune response against the tumor. Autologous TILs are engineered to equip them with activity against TAAs using modified TCRs or Chimeric Antigen Receptors (CARs). CARs are recombinant receptors with an intracellular signaling portion constituting T-cell receptor–CD3-ζ complex and an extracellular single chain variable fragment of mAb [Gross et al. 1989; Khan et al. 2014]. The first generation CARs have FcRγ or CD3ζ chains and second-generation CARs incorporates additional cytoplasmic costimulatory domains such as CD28 and CD137 and third-generation CARs comprise three signaling domains. This helps T cells to target tumor cells in a HLA-independent manner to a wide range of surface TAAs [Kershaw et al. 1996].

A preclinical study on murine model expressing anti-CEA CAR T cells were able to reduce the size of pancreatic tumors below the limit of detection and long-term tumor eradication in 67% of mice [Chmielewski et al. 2012]. Intratumoral injection of T cells expressing CAR against Her2/neu or CD24 completely eliminated the tumor and more than 50% of animals appeared to be disease-free more than 2 months later [Maliar et al. 2012]. Similarly other studies with T cells expressing MUC-1 CAR T cells plus IL-4 receptor α [Wilkie et al. 2010] and mesothelin CAR T cells [Carpenito et al. 2009, Zhao et al. 2010] resulted in tumor regression in large, pre-established tumors in mice.

MORAb-009 is a chimeric IgG1 antibody targeting tumor-associated mesothelin overexpressed on pancreatic, ovarian, lung and colorectal carcinoma. A preclinical evaluation of MORAb-900 found the increased effectiveness that led to reduction in tumor growth of mesothelin positive tumors when combined with chemotherapy [Hassan et al. 2007]. This was tested further in a phase I clinical study [Hassan et al. 2010] in 24 mesothelin positive patients including 7 pancreatic cancer patients which showed it was safe and well tolerated at a dose of 200 mg/m². A total of 11 of previously treated patients in this group had stable disease. Recently reported results of randomized phase II clinical trial [ClinicalTrials.gov identifier: NCT00570713] of MORAb-009 in combination with gemcitabine or placebo in 155 advanced pancreatic cancer subjects failed to suggest any significant benefit of MORAb-900 therapy. The OS of MORAb arm was found to be 6.5 months (4.5–8.10) versus 6.9 months (5.4–8.8) in the placebo arm and PFS of 3.4 months (1.9–4.7) and 3.5 (2.8–4.9), respectively. A total of 19.2% of the patients had progressive disease in the MORAb-009 arm as compared with 23.4% patients in the placebo arm.

Despite the results of the above-mentioned trials, majority of patients has failed to respond well to ACT. Tumor-induced immunosuppression, i.e. upregulation of inhibitory receptors and ligands of activated T cells have shown to contribute to failure of T-cell therapy [Abate-Daga et al. 2013]. To overcome these inhibitory signals, Kobold and colleagues developed a new fusion PD-1 – CD28 receptor hypothesizing that CD28 costimulation may overcome peripheral tolerance [Kobold et al. 2015]. Treatment of mice with Panc02 pancreatic carcinoma cell line expressing ovalbumin (Panc-OVA) with PD-1–CD28 fusion receptor led to complete regression of tumor and had a memory response in 9 out of 11 mice. It warrants further development of the strategy to enhance efficacy of ACT in cancer. Another approach tested preclinically to improve the activity of antitumor CAR T cells is through increased constitutive expression of CD40L. T cells demonstrated increased proliferation and secretion of proinflammatory Th1 cytokines and increased CD19-specific CAR/CD40L T cells cytotoxicity against tumor cells. It also augmented immunogenicity of CD40+ tumor cells by upregulated expression of various costimulatory, adhesion molecules thereby counteracting the tumor cells ability for immune evasion. These novel approaches should be tested in additional studies with the goal of ascertaining their usefulness in a clinical setting. Completed and ongoing studies with adoptive T-cell therapy in pancreatic cancer alone or in solid tumor cancer groups including pancreatic cancer are depicted in Table 12.

Table 12.

Adoptive T-cell therapy in clinical trials.

Intervention	Strategy	Date (last updated)	Status	Patient population	Phase	Study design	ClinicalTrials.gov identifier
Autologous T-cells with chimeric anti-mesothelin SS1 receptor	Mesothelin RNA CAR T-cell therapy	8 January 2015	Completed	Metastatic PDAC	I	I.v. administration of intervention to study safety and feasibility	NCT01897415
CART-meso +/– Cyclophosphamide	Mesothelin CAR T-cell therapy.Cytotoxic drugs	30 November 2015	Recruiting	Mesothelin expressing cancers including metastatic PDAC	I	Dose escalation design transducing CART-meso cells with or without CY	NCT02159716
Anti-mesothelin CAR, Cyclophosphamide, Fludarabine,Aldesleukin	CAR T cell therapy.Cytotoxic drugs.IL-2 cytokine	3 November 2015	Recruiting	Metastatic pancreatic cancer	I/II	Single arm with infusion of lymphodepleting cytotoxic drugs followed by CART-meso + Aldesleukin	NCT01583686
Anti-NY ESO-1 mTCR PBL, Cyclophosphamide, Fludarabine, Aldesleukin	Anti-NY-ESO-1 murine TCR peripheral blood lymphocyte.Cytotoxic drugs.IL-2 cytokine	7 November 2015	Recruiting	Metastatic cancer expressing NY-ESO-1 antigen	II	Single arm with infusion of lymphodepleting cytotoxic drugs followed by anti-NY-ESO-1 mTCR PBL and high-dose aldesleukin	NCT01967823
Anti-MAGE-A3-DP4 TCR PBL, Cyclophosphamide, Fludarabine, Aldesleukin	Anti-melanoma antigen-3 reactive T-cell therapy.Cytotoxic drugs.IL-2 cytokine	22 September 2015	Recruiting	Metastatic cancer expressing the MAGE-A3-DP4 antigen	I/II	Single arm with infusion of lymphodepleting cytotoxic drugs followed by anti-MAGE-A3-DP4 TCR PBL plus aldesleukin	NCT02111850
Anti VEGFR2 TCR, Cyclophosphamide,Fludarabine, Aldesleukin	Anti-VEGFR2 reactive T-cell therapy.Cytotoxic drugs.IL-2 cytokine	30 September 2015	Ongoing but not recruiting	Metastatic cancer with evaluable disease	I/II	Single arm with infusion of lymphodepleting cytotoxic drugs followed by anti-VEGFR2 TCR plus aldesleukin	NCT01218867
Anti-CEA-CAR T	Carcinogenic embryonic antigen CAR T cell therapy	21 June 2015	Recruiting	CEA positive cancer	I	Efficacy study	NCT02349724
Anti-CD133-CAR vector-transduced T cells	CAR T cell therapy	2 September 2015	Recruiting	Relapsed or chemotherapy refractory malignancies	I	Dose-escalation study	NCT02541370
CART-meso-19 T cells, Cyclophosphamide	CAR T cell therapy,Cytotoxic drugs.	20 July 2015	Recruiting	Metastatic pancreatic cancer.	I	Cyclophosphamide followed by single dose of combined CART-meso and CART-19 cells.	NCT02465983
Young TILAldesleukinCyclophosphamideFludarabine	Autologous transfer of tumor infiltrating lymphocytes,Cytotoxic drugs	26 November 2015	Recruiting	Metastatic cancer including pancreatic cancer	II	Lymphodepleting drugs followed by i.v. young TILs PBL and aldesleukin	NCT01174121

Immune modulating agents

CD40 agonist antibodies

CD40 is a cell surface receptor that belongs to the family of tumor necrosis factor receptor and is expressed on various APCs including DCs, monocytes, macrophages, mast cells etc. and its ligand CD40L (CD154) is a type II transmembrane protein predominantly expressed on activated T cells. CD40–CD40L interactions provide proliferative signals to promote differentiation of CD4+ and CD8+ T cells and hence may play a role in upregulation of immunity [Grewal and Flavell, 1998; Van Kooten and Banchereau, 2000]. As described earlier, pancreatic cancer leads to immune suppression within the tumor environment by inhibiting costimulatory molecules and also via upregulation of inhibitory molecules on inactivated T cells. Activation of CD40 has shown to restrict tumor growth in vitro and in vivo in many preclinical studies [Funakoshi et al. 1994; Eliopoulos et al. 2000; Tong et al. 2001]. Clinical activity of CD40 molecule was studied by giving CP-870,893, a fully human and selective CD40 agonist mAb in 29 patients with advanced solid tumors. A partial response was observed in four patients with metastatic melanoma and dose-related upregulation of costimulatory molecules on B cells in peripheral blood was also noted [Vonderheide et al. 2007].

Pancreatic cancer tissues have a higher expression of CD40 as compared to adjacent normal tissues. In a study by He and colleagues [He et al. 2012] 69% patients had a positive expression of CD40 and it correlated with higher TNM staging and lymph node metastasis. Also there were increased serum sCD40L levels compared with the healthy subjects. Recombinant CD40L leading to activation of CD40 significantly inhibited the proliferation of pancreatic cancer cell lines and induction of apoptosis. In a phase I study patients with advanced solid tumors were infused weekly doses of CP-870,893 which were well tolerated but there was little clinical activity [Rüter et al. 2010]. A combination of CD40 agonist antibody was tested in combination with gemcitabine in genetically engineered mouse model of PDAC. There was a depletion of CD40+ macrophages in stroma which infiltrated the tumor and became tumoricidal suggesting the CD40+ T-cell-independent mechanism of tumor regression [Beatty et al. 2011]. This was subsequently tested in clinical study. Beatty and colleagues investigated maximum tolerated dose, safety and antitumor activity of CP-870,893 with gemcitabine combination in 22 patients with advanced PDAC. Using RECIST criteria, four patients had a partial response, 11 with stable disease with PET demonstrating more than 25% decrease in FDG uptake within primary pancreatic lesions. One patient with partial response also had a complete regression of 7.6 cm liver mass and 47% regression of a second liver metastasis. Increase in B-cell expansion of costimulatory molecules was seen along with markedly increased inflammatory cytokines. PFS of 5.6 months and OS of 7.4 months was promising. Improved OS correlated with a decrease in FDG uptake in hepatic lesions suggesting the early role of FDG-PET/CT imaging [Beatty et al. 2013]. Table 13 summarizes completed and ongoing studies with CD40 agonist antibodies.

Table 13.

Studies using CD40 agonist antibody.

Intervention	Strategy	Patient population	Phase	Year	Results	ClinicalTrials.gov identifier
CP-870,893 + Gemcitabine.	Monoclonal antibody and cytotoxic drugs	Pancreatic neoplasms	I	2013	Overall survival 8.4 months Progression-free survival 5.3 months	NCT00711191
CP-870,893 + Gemcitabine	Monoclonal antibody and cytotoxic drugs	PDAC	I	2014	Pending	NCT01456585
CP-870,893	Monoclonal antibody	Advanced solid tumors	I	2014	Pending	NCT02225002

(C-C motif) receptor 2 inhibitor

CCR2 is a chemokine receptor which mediates chemotaxis of immune cells and is expressed on monocytes and macrophages. Chronic tumor infiltrating cells might play a role in tumor progression, metastasis and resistance to chemotherapy. Targeting tumor-associated macrophages (TAMs) by inhibiting CCR2 would decrease the immune cells in tumor environment and increases efficacy of chemotherapy [Mitchem et al. 2013]. PF-04136309 is a novel CCR2 inhibitor with preclinical antitumor activity in PDAC through depleting TAMs and inflammatory monocytes. A phase Ib study of PF-04136309 given with FOLFIRINOX in 41 patients was conducted. A total of 12 of 23 evaluable patients (52.2%) had partial response by RECIST and 11 (47.8%) had stable disease. Curative resection was achieved in four out of five patients with borderline resectable and two with locally advanced disease [Wang-Gillam et al. 2015]. The data thus far are promising and need to be further validated.

Conclusion

Immunotherapy is a promising emerging treatment in cancer care. Manipulation of the immune targets along with traditional chemotherapy displays a synergy in antitumor response suggesting a probable role for immunotherapy as an adjuvant or in combination with radiochemotherapy. Furthermore, combining different immunotherapeutic strategies has shown some favorable outcomes with significant clinical benefits. Additional larger clinical trials testing the complex of immune checkpoint inhibitors with use of one or more types of vaccines and newer personalized therapies are warranted. Pancreatic cancer is a systemic disease which remains highly lethal even when resected. Therefore, early administration of effective combination therapy including immunotherapy may prove beneficial and needs further evaluation.

Footnotes

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Conflict of interest statement

The authors declare that there is no conflict of interest.

References

Abate-Daga

Hanada

Davis

Yang

Rosenberg

Morgan

(2013) Expression profiling of TCR-engineered T cells demonstrates overexpression of multiple inhibitory receptors in persisting lymphocytes. Blood 122: 1399–1410.

Abou-Alfa

Chapman

Feilchenfeldt

Brennan

Capanu

Gansukh

. (2011) Targeting mutated K-Ras in pancreatic adenocarcinoma using an adjuvant vaccine. Am J Clin Oncol 34: 321–325.

Abrams

Khleif

Bergmann-Leitner

Kantor

Chung

Hamilton

. (1997) Generation of stable CD4+and CD8+T cell lines from patients immunized with Ras oncogene-derived peptides reflecting codon 12 mutations. Cell Immunol 182: 137–151.

Amedei

Niccolai

Benagiano

Della Bella

Cianchi

Bechi

. (2013) Ex vivo analysis of pancreatic cancer-infiltrating T lymphocytes reveals that ENO-specific Tregs accumulate in tumor tissue and inhibit Th1/Th17 effector cell functions. Cancer Immunol Immunother 62: 1249–1260.

Bafna

Kaur

Momi

Batra

(2009) Pancreatic cancer cells resistance to gemcitabine: the role of MUC4 mucin. Br J Cancer 101: 1155–1161.

Beatty

Chiorean

Fishman

Saboury

Teitelbaum

Sun

. (2011) CD40 agonists alter tumor stroma and show efficacy against pancreatic carcinoma in mice and humans. Science 331: 1612–1616.

Beatty

Torigian

Chiorean

Saboury

Brothers

Alavi

. (2013) A phase I study of an agonist CD40 monoclonal antibody (CP-870,893) in combination with gemcitabine in patients with advanced pancreatic ductal adenocarcinoma. Clin Cancer Res 19: 6286–6295.

Behrens

Grandgenett

Bailey

Singh

. (2010) The reactive tumor microenvironment: MUC1 signaling directly reprograms transcription of CTGF. Oncogene 29: 5667–5677.

Bernhardt

Gjertsen

Trachsel

Møller

Eriksen

Meo

. (2006) Telomerase peptide vaccination of patients with non-resectable pancreatic cancer: a dose escalating phase I/II study. Br J Cancer 95: 1474–1482.

10.

Besmer

Curry

Roy

Tinder

Sahraei

Schettini

. (2011) Pancreatic ductal adenocarcinoma mice lacking mucin 1 have a profound defect in tumor growth and metastasis. Cancer Res 71: 4432–4442.

11.

Birmingham

Canadien

Kaniuk

Steinberg

Higgins

Brumell

(2008) Listeriolysin O allows Listeria monocytogenes replication in macrophage vacuoles. Nature 451: 350–354.

12.

Blank

Enk

(2015) Therapeutic use of anti-CTLA-4 antibodies. Int Immunol 27: 3–10.

13.

Brahmer

Tykodi

Chow

Hwu

Topalian

Hwu

. (2012) Safety and activity of anti-PD-L1 antibody in patients with advanced cancer. N Engl J Med 366: 2455–2465.

14.

Brett

Smith

Bouvier

Michaeli

Hochhauser

Davidson

. (2002) Phase II study of anti-gastrin-17 antibodies, raised to G17DT, in advanced pancreatic cancer. J Clin Oncol 20: 4225–4231.

15.

Brunet

Denizot

Luciani

Roux-Dosseto

Suzan

Mattei

. (1987) A new member of the immunoglobulin superfamily—CTLA-4. Nature 328: 267–270.

16.

Buanes

Maurel

Liauw

Hebbar

Nemunaitis

(2009) A randomized phase III study of gemcitabine (G) versus GV1001 in sequential combination with G in patients with unresectable and metastatic pancreatic cancer (PC). ASCO Meeting Abstracts 27: 4601.

17.

Burdick

Harris

Reid

Iwamura

Hollingsworth

(1997) Oligosaccharides expressed on MUC1 produced by pancreatic and colon tumor cell lines. J Biol Chem 272: 24198–24202.

18.

Calabrò

Morra

Fonsatti

Cutaia

Amato

Giannarelli

. (2013) Tremelimumab for patients with chemotherapy-resistant advanced malignant mesothelioma: an open-label, single-arm, phase II trial. Lancet Oncol 14: 1104–1111.

19.

Carbone

Ciernik

Kelley

Smith

Nadaf

Kavanaugh

. (2005) Immunization with mutant p53- and K-Ras–derived peptides in cancer patients: immune response and clinical outcome. J Clin Oncol 23: 5099–5107.

20.

Carpenito

Milone

Hassan

Simonet

Lakhal

Suhoski

. (2009) Control of large, established tumor xenografts with genetically retargeted human T cells containing CD28 and CD137 domains. Proc Natl Acad Sci U S A 106: 3360–3365.

21.

Cereda

Vergati

Huen

Di Bari

Jochems

Intrivici

. (2011) Maturation of human dendritic cells with Saccharomyces cerevisiae (yeast) reduces the number and function of regulatory T cells and enhances the ratio of antigen-specific effectors to regulatory T cells. Vaccine 29: 4992–4999.

22.

Chen

Flies

(2013) Molecular mechanisms of T cell co-stimulation and co-inhibition. Nat Rev Immunol 13: 227–242.

23.

Chmielewski

Hahn

Rappl

Nowak

Schmidt–Wolf

Hombach

. (2012) T cells that target carcinoembryonic antigen eradicate orthotopic pancreatic carcinomas without inducing autoimmune colitis in mice. Gastroenterology 143: 1095–1107. e1092. doi: 10.1053/j.gastro.2012.06.037.

24.

Clark

Hingorani

Mick

Combs

Tuveson

Vonderheide

(2007) Dynamics of the immune reaction to pancreatic cancer from inception to invasion. Cancer Res 67: 9518–9527.

25.

Conroy

Desseigne

Ychou

Bouché

Guimbaud

Bécouarn

. (2011) FOLFIRINOX versus gemcitabine for metastatic pancreatic cancer. N Engl J Med 364: 1817–1825.

26.

Counter

Meyerson

Eaton

Ellisen

Caddle

Haber

. (1998) Telomerase activity is restored in human cells by ectopic expression of hTERT (hEST2), the catalytic subunit of telomerase. Oncogene 16: 1217–1222.

27.

Davis

Conlon

Bohac

Barcenas

Leslie

Watkins

. (2012) Effect of pemetrexed on innate immune killer cells and adaptive immune T cells in subjects with adenocarcinoma of the pancreas. J Immunother 35: 629–640.

28.

De Remigis

De Gruijl

Uram

Tzou

Iwama

Talor

. (2015) Development of thyroglobulin antibodies after GVAX immunotherapy is associated with prolonged survival. Int J Cancer 136: 127–137.

29.

Dong

Qian

Wang

Meng

Chen

. (2015) Survivin expression and serum levels in pancreatic cancer. World J Surg Oncol 13: 189.

30.

Dong

Strome

Salomao

Tamura

Hirano

Flies

. (2002) Tumor-associated B7-H1 promotes T-cell apoptosis: a potential mechanism of immune evasion. Nat Med 8: 793–800.

31.

Eliopoulos

Davies

Knox

Gallagher

Afford

Adams

. (2000) CD40 induces apoptosis in carcinoma cells through activation of cytotoxic ligands of the tumor necrosis factor superfamily. Mol Cell Biol 20: 5503–5515.

32.

Ferlay

Soerjomataram

Dikshit

Eser

Mathers

Rebelo

. (2015) Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer 136: E359–E386.

33.

Francisco

Sage

Sharpe

(2010) The PD-1 pathway in tolerance and autoimmunity. Immunol Rev 236: 219–242.

34.

Funakoshi

Longo

Beckwith

Conley

Tsarfaty

. (1994) Inhibition of human B-cell lymphoma growth by CD40 stimulation. Blood 83: 2787–2794.

35.

Galili

Macher

Buehler

Shohet

(1985) Human natural anti-alpha-galactosyl IgG. II. The specific recognition of alpha (1—3)-linked galactose residues. J Exp Med 162: 573–582.

36.

Galili

Mandrell

Hamadeh

Shohet

Griffiss

(1988) Interaction between human natural anti-alpha-galactosyl immunoglobulin G and bacteria of the human flora. Infect Immun 56: 1730–1737.

37.

Gaudernack

(2006) Prospects for vaccine therapy for pancreatic cancer. Best Pract Res Clin Gastroenterol 20: 299–314.

38.

Geng

Huang

Liu

Qian

Deng

. (2008) B7-H1 up-regulated expression in human pancreatic carcinoma tissue associates with tumor progression. J Cancer Res Clin Oncol 134: 1021–1027.

39.

Gilliam

Broome

Topuzov

Avgust

Pulay

Humphreys

. (2012) An international multicenter randomized controlled trial of G17DT in patients with pancreatic cancer. Pancreas 41: 374–379.

40.

Gilliam

Topuzov

Garin

Pulay

Broome

Watson

. (2004) Randomised, double blind, placebo-controlled, multi-centre, group-sequential trial of G17DT for patients with advanced pancreatic cancer unsuitable or unwilling to take chemotherapy. ASCO Meeting Abstracts 22: 2511.

41.

Gjertsen

Bakka

Breivik

Saeterdal

Solheim

Søreide

. (1995) Vaccination with mutant ras peptides and induction of T-cell responsiveness in pancreatic carcinoma patients carrying the corresponding RAS mutation. Lancet 346: 1399–1400.

42.

Gjertsen

Bjørheim

Saeterdal

Myklebust

Gaudernack

(1997) Cytotoxic CD4+ and CD8+ T lymphocytes, generated by mutant p21-ras (12Val) peptide vaccination of a patient, recognize 12Val-dependent nested epitopes present within the vaccine peptide and kill autologous tumour cells carrying this mutation. Int J Cancer 72: 784–790.

43.

Gjertsen

Buanes

Rosseland

Bakka

Gladhaug

Søreide

. (2001) Intradermal ras peptide vaccination with granulocyte-macrophage colony-stimulating factor as adjuvant: clinical and immunological responses in patients with pancreatic adenocarcinoma. Int J Cancer 92: 441–450.

44.

Goldstein

El-Maraghi

Hammel

Heinemann

Kunzmann

Sastre

. (2015) nab-Paclitaxel plus gemcitabine for metastatic pancreatic cancer: long-term survival from a phase III trial. J Natl Cancer Inst 107: dju413. doi:10.1093/jnci/dju413.

45.

Gorelik

Duty

Anaraki

Galili

(1995) Alterations of cell surface carbohydrates and inhibition of metastatic property of murine melanomas by α1,3 galactosyltransferase gene transfection. Cancer Res 55: 4168–4173.

46.

Goydos

Elder

Whiteside

Finn

Lotze

(1996) A phase I trial of a synthetic mucin peptide vaccine: induction of specific immune reactivity in patients with adenocarcinoma. J Surg Res 63: 298–304.

47.

Grewal

Flavell

(1998) CD40 and CD154 in cell-mediated immunity. Annu Rev Immunol 16: 111–135.

48.

Gross

Waks

Eshhar

(1989) Expression of immunoglobulin-T-cell receptor chimeric molecules as functional receptors with antibody-type specificity. Proc Natl Acad Sci U S A 86: 10024–10028.

49.

Hardacre

Mulcahy

Small

Talamonti

Obel

Rocha Lima

. (2012) Addition of algenpantucel-L immunotherapy to standard of care (SOC) adjuvant therapy for pancreatic cancer. ASCO Meeting Abstracts 30: 4049.

50.

Hassan

Cohen

Phillips

Pastan

Sharon

Kelly

. (2010) Phase I clinical trial of the chimeric anti-mesothelin monoclonal antibody MORAB-009 in patients with mesothelin expressing cancers. Clin Cancer Res 16: 6132–6138.

51.

Hassan

Ebel

Routhier

Patel

Kline

Zhang

. (2007) Preclinical evaluation of MORAb-009, a chimeric antibody targeting tumor-associated mesothelin. Cancer Immun 7: 20.

52.

Zhao

Fei

Wang

Zhu

. (2012) Expression of the co-signaling molecules CD40-CD40l and their growth inhibitory effect on pancreatic cancer in vitro. Oncol Rep 28: 262–268.

53.

Herbst

Soria

Kowanetz

Fine

Hamid

Gordon

. (2014) Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancer patients. Nature 515: 563–567.

54.

Hodi

O’day

Mcdermott

Weber

Sosman

Haanen

. (2010) Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J Med 363: 711–723.

55.

Howlader

Noone

Krapcho

Garshell

Miller

Altekruse

. (2015) Seer Cancer Statistics Review, 1975–2012. Bethesda, MD: National Cancer Institute.

56.

Hruban

Van Mansfeld

Offerhaus

Van Weering

Allison

Goodman

. (1993) K-ras oncogene activation in adenocarcinoma of the human pancreas. A study of 82 carcinomas using a combination of mutant-enriched polymerase chain reaction analysis and allele-specific oligonucleotide hybridization. Am J Pathol 143: 545–554.

57.

Huang

Golumbek

Ahmadzadeh

Jaffee

Pardoll

Levitsky

(1994) Role of bone marrow-derived cells in presenting MHD class I-restricted tumor antigens. Science 264: 961–965.

58.

Ino

Yamazaki-Itoh

Shimada

Iwasaki

Kosuge

Kanai

. (2013) Immune cell infiltration as an indicator of the immune microenvironment of pancreatic cancer. Br J Cancer 108: 914–923.

59.

Ishizaki

Manuel

Song

Srivastava

Sun

Diamond

. (2011) Modified vaccinia Ankara expressing survivin combined with gemcitabine generates specific antitumor effects in a murine pancreatic carcinoma model. Cancer Immunol Immunother 60: 99–109.

60.

Iwai

Ishida

Tanaka

Okazaki

Honjo

Minato

(2002) Involvement of PD-L1 on tumor cells in the escape from host immune system and tumor immunotherapy by PD-L1 blockade. Proc Natl Acad Sci U S A 99: 12293–12297.

61.

Jaffee

Hruban

Biedrzycki

Laheru

Schepers

Sauter

. (2001) Novel allogeneic granulocyte-macrophage colony-stimulating factor-secreting tumor vaccine for pancreatic cancer: a phase I trial of safety and immune activation. J Clin Oncol 19: 145–156.

62.

Kami

Doi

Koizumi

Toyoda

Mori

Ito

. (2004) Survivin expression is a prognostic marker in pancreatic cancer patients. Surgery 136: 443–448.

63.

Kershaw

Darcy

Trapani

Smyth

(1996) The use of chimeric human Fc(epsilon) receptor I to redirect cytotoxic T lymphocytes to tumors. J Leukoc Biol 60: 721–728.

64.

Khan

Halfdanarson

Borad

(2014) Immunotherapeutic and oncolytic viral therapeutic strategies in pancreatic cancer. Future Oncol 10: 1255–1275.

65.

Kirkwood

Lorigan

Hersey

Hauschild

Robert

Mcdermott

. (2010) Phase II trial of tremelimumab (CP-675,206) in patients with advanced refractory or relapsed melanoma. Clin Cancer Res 16: 1042–1048.

66.

Kobold

Grassmann

Chaloupka

Lampert

Wenk

Kraus

. (2015) Impact of a new fusion receptor on PD-1–mediated immunosuppression in adoptive T cell therapy. J Natl Cancer Inst 107: djv146. doi:10.1093/jnci/djv146.

67.

Koido

Hara

Homma

Torii

Toyama

Kawahara

. (2005) Dendritic cells fused with allogeneic colorectal cancer cell line present multiple colorectal cancer-specific antigens and induce antitumor immunity against autologous tumor cells. Clin Cancer Res 11: 7891–7900.

68.

Kondo

Hazama

Kawaoka

Yoshino

Yoshida

Tokuno

. (2008) Adoptive immunotherapy for pancreatic cancer using MUC1 peptide-pulsed dendritic cells and activated T lymphocytes. Anticancer Res 28: 379–387.

69.

Koski

Cohen

Roses

Czerniecki

(2008) Reengineering dendritic cell-based anti-cancer vaccines. Immunol Rev 222: 256–276.

70.

Laheru

Lutz

Burke

Biedrzycki

Solt

Onners

. (2008) Allogeneic GM-CSF secreting tumor immunotherapy (Gvax^®) alone or in sequence with cyclophosphamide for metastatic pancreatic cancer: a pilot study of safety, feasibility and immune activation. Clin Cancer Res 14: 1455–1463.

71.

Brockstedt

Nir-Paz

Hampl

Mathur

Nemunaitis

. (2012) A live-attenuated Listeria vaccine (ANZ-100) and a live-attenuated Listeria vaccine expressing mesothelin (CRS-207) for advanced cancers: phase 1 studies of safety and immune induction. Clin Cancer Res 18: 858–868.

72.

Lutz

Uram

Sugar

Onners

Solt

. (2013) Evaluation of ipilimumab in combination with allogeneic pancreatic tumor cells transfected with a GM-CSF gene in previously treated pancreatic cancer. J Immunother 36: 382–389.

73.

Wang-Gillam

Picozzi

Greten

Crocenzi

Springett

. (2015) Safety and survival with GVAX pancreas prime and Listeria Monocytogenes–expressing mesothelin (CRS-207) boost vaccines for metastatic pancreatic cancer. J Clin Oncol 33: 1325–1333.

74.

Leach

Krummel

Allison

(1996) Enhancement of antitumor immunity by CTLA-4 blockade. Science 271: 1734–1736.

75.

Lepisto

Moser

Zeh

Lee

Bartlett

Mckolanis

. (2008) 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 Ther 6: 955–964.

76.

Liyanage

Moore

Joo

Tanaka

Herrmann

Doherty

. (2002) Prevalence of regulatory T cells is increased in peripheral blood and tumor microenvironment of patients with pancreas or breast adenocarcinoma. J Immunol 169: 2756–2761.

77.

Lutz

Yeo

Lillemoe

Biedrzycki

Kobrin

Herman

. (2011) A lethally irradiated allogeneic granulocyte-macrophage colony stimulating factor-secreting tumor vaccine for pancreatic adenocarcinoma: a phase II trial of safety, efficacy, and immune activation. Ann Surg 253: 328–335.

78.

Mahoney

Freeman

McDermott

(2015) The next immune-checkpoint inhibitors: PD-1/PD-L1 blockade in melanoma. Clin Ther 37: 764–782.

79.

Maki

Livingston

Lewis

Janetzki

Klimstra

Desantis

. (2007) A phase I pilot study of autologous heat shock protein vaccine HSPPC-96 in patients with resected pancreatic adenocarcinoma. Dig Dis Sci 52: 1964–1972.

80.

Maliar

Servais

Waks

Chmielewski

Lavy

Altevogt

. (2012) Redirected T cells that target pancreatic adenocarcinoma antigens eliminate tumors and metastases in mice. Gastroenterology 143: 1375–1384. e1-5. doi:10.1053/j.gastro.2012.07.017.

81.

Mandell

Flick

Staplin

Kaniewski

Carzoli

Manuszak

. (2009) The αGal HyperAcute® Technology: enhancing immunogenicity of antiviral vaccines by exploiting the natural αGal-mediated zoonotic blockade. Zoonoses Public Health 56: 391–406.

82.

Mazzolini

Alfaro

Sangro

Feijoó

Ruiz

Benito

. (2005) Intratumoral injection of dendritic cells engineered to secrete interleukin-12 by recombinant adenovirus in patients with metastatic gastrointestinal carcinomas. J Clin Oncol 23: 999–1010.

83.

Middleton

Valle

Wadsley

Propper

Coxon

Ross

. (2013) A phase III randomized trial of chemoimmunotherapy comprising gemcitabine and capecitabine with or without telomerase vaccine GV1001 in patients with locally advanced or metastatic pancreatic cancer. ASCO Meeting Abstracts 31: LBA4004.

84.

Mitchem

Brennan

Knolhoff

Belt

Zhu

Sanford

. (2013) Targeting tumor-infiltrating macrophages decreases tumor-initiating cells, relieves immunosuppression and improves chemotherapeutic responses. Cancer Res 73: 1128–1141.

85.

Miyazawa

Ohsawa

Tsunoda

Hirono

Kawai

Tani

. (2010) 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 Sci 101: 433–439.

86.

Morse

Nair

Boczkowski

Tyler

Hurwitz

Proia

. (2002) The feasibility and safety of immunotherapy with dendritic cells loaded with CEA mRNA following neoadjuvant chemoradiotherapy and resection of pancreatic cancer. Int J Gastrointest Cancer 32: 1–6.

87.

Nomi

Sho

Akahori

Hamada

Kubo

Kanehiro

. (2007) Clinical significance and therapeutic potential of the programmed death-1 ligand/programmed death-1 pathway in human pancreatic cancer. Clin Cancer Res 13: 2151–2157.

88.

Oettle

Richards

Ramanathan

Van Laethem

Peeters

Fuchs

. (2005) A phase III trial of pemetrexed plus gemcitabine versus gemcitabine in patients with unresectable or metastatic pancreatic cancer. Ann Oncol 16: 1639–1645.

89.

Ogata

Naito

Tanaka

Moriyama

Asano

(2000) Overexpression and localization of heat shock proteins mRNA in pancreatic carcinoma. J Nippon Med Sch 67: 177–185.

90.

Okudaira

Hokari

Tsuzuki

Okada

Komoto

Watanabe

. (2009) Blockade of B7-H1 or B7-DC induces an anti-tumor effect in a mouse pancreatic cancer model. International Journal of Oncology 35: 741–749.

91.

Palucka

Banchereau

(2013) Dendritic cell-based cancer therapeutic vaccines. Immunity 39: 38–48.

92.

Pardoll

(2012) The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer 12: 252–264.

93.

Paterson

Guirnalda

Wood

(2010) Listeria and Salmonella bacterial vectors of tumor-associated antigens for cancer immunotherapy. Semin Immunol 22: 183–189.

94.

Pecher

Häring

Kaiser

Thiel

(2002) Mucin gene (MUC1) transfected dendritic cells as vaccine: results of a phase I/II clinical trial. Cancer Immunol Immunother 51: 669–673.

95.

Pedoeem

Azoulay-Alfaguter

Strazza

Silverman

Mor

(2014) Programmed death-1 pathway in cancer and autoimmunity. Clin Immunol 153: 145–152.

96.

Peggs

Quezada

Chambers

Korman

Allison

(2009) Blockade of CTLA-4 on both effector and regulatory T cell compartments contributes to the antitumor activity of anti–CTLA-4 antibodies. J Exp Med 206: 1717–1725.

97.

Plate

Shott

Bograd

Harris

(2005) Effect of gemcitabine on immune cells in subjects with adenocarcinoma of the pancreas. Cancer Immunol Immunother 54: 915–925.

98.

Song

Rizvi

Raja

Zhang

. (2004) MUC1 expression in primary and metastatic pancreatic cancer cells for in vitro treatment by (213)Bi-C595 radioimmunoconjugate. Br J Cancer 91: 2086–2093.

99.

Ramanathan

Lee

Mckolanis

Hitbold

Schraut

Moser

. (2005) 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 Immunol Immunother 54: 254–264.

100.

Razzaque

Ashraf

Chavez

Malafa

Coppola

Springett

. (2013) Expression of programmed death ligand 1 (PD-L1) in malignant and nonmalignant pancreatic tissue. ASCO Meeting Abstracts 31: 215.

101.

Ribas

Hanson

Noe

Millham

Guyot

Bernstein

. (2007) Tremelimumab (CP-675,206), a cytotoxic T lymphocyte–associated antigen 4 blocking monoclonal antibody in clinical development for patients with cancer. Oncologist 12: 873–883.

102.

Ribas

Kefford

Marshall

Punt

Haanen

Marmol

. (2013) Phase III randomized clinical trial comparing tremelimumab with standard-of-care chemotherapy in patients with advanced melanoma. J Clin Oncol 31: 616–622.

103.

Robert

Ribas

Wolchok

Hodi

Hamid

Kefford

. (2014) Anti-programmed-death-receptor-1 treatment with pembrolizumab in ipilimumab-refractory advanced melanoma: a randomised dose-comparison cohort of a phase I trial. Lancet 384: 1109–1117.

104.

Rosenwald

Wright

Leroy

Gaulard

Gascoyne

. (2003) Molecular diagnosis of primary mediastinal B cell lymphoma identifies a clinically favorable subgroup of diffuse large B cell lymphoma related to Hodgkin lymphoma. J Exp Med 198: 851–862.

105.

Rossi

Mautino

Unfer

Seregina

Vahanian

Link

(2005) Effective treatment of preexisting melanoma with whole cell vaccines expressing α(1,3)-galactosyl epitopes. Cancer Res 65: 10555–10561.

106.

Royal

Levy

Turner

Mathur

Hughes

Kammula

. (2010) Phase II trial of single agent ipilimumab (anti-CTLA-4) for locally advanced or metastatic pancreatic adenocarcinoma. J Immunother 33: 828–833.

107.

Rudd

(2008) The reverse stop-signal model for CTLA4 function. Nat Rev Immunol 8: 153–160.

108.

Rüter

Antonia

Burris

Huhn

Vonderheide

(2010) Immune modulation with weekly dosing of an agonist CD40 antibody in a phase I study of patients with advanced solid tumors. Cancer Biol Ther 10: 983–993.

109.

Ryan

O’Donovan

Duffy

(2009) Survivin: a new target for anti-cancer therapy. Cancer Treat Rev 35: 553–562.

110.

Schmidt

Ziske

Märten

Endres

Tiemann

Schmitz

. (2003) Intratumoral immunization with tumor RNA-pulsed dendritic cells confers antitumor immunity in a C57BL/6 pancreatic murine tumor model. Cancer Res 63: 8962–8967.

111.

Schneider

Downey

Smith

Zinselmeyer

Rush

Brewer

. (2006) Reversal of the TCR stop signal by CTLA-4. Science 313: 1972–1975.

112.

Shapiro

Marshall

Karasek

Figer

Oettle

Couture

. (2005) G17DT+gemcitabine [Gem] versus placebo+Gem in untreated subjects with locally advanced, recurrent, or metastatic adenocarcinoma of the pancreas: results of a randomized, double-blind, multinational, multicenter study. ASCO Meeting Abstracts 23: LBA4012.

113.

Singh

Dominiecki

Jaffee

Paterson

(2005) Fusion to Listeriolysin O and delivery by Listeria monocytogenes enhances the immunogenicity of HER-2/neu and reveals subdominant epitopes in the FVB/N mouse. J Immunol 175: 3663–3673.

114.

Skrypek

Duchêne

Hebbar

Leteurtre

Van Seuningen

Jonckheere

(2013) The MUC4 mucin mediates gemcitabine resistance of human pancreatic cancer cells via the Concentrative Nucleoside Transporter family. Oncogene 32: 1714–1723.

115.

Song

Liu

Jin

Huang

(2014) Overexpression of B7-H1 correlates with malignant cell proliferation in pancreatic cancer. Oncol Rep 31: 1191–1198.

116.

Dannull

Yang

Dahm

Coleman

Yancey

. (2005) Telomerase mRNA-transfected dendritic cells stimulate antigen-specific CD8+ and CD4+ T cell responses in patients with metastatic prostate cancer. J Immunol 174: 3798–3807.

117.

Suehara

Mizumoto

Muta

Tominaga

Shimura

Kitajima

. (1997) Telomerase elevation in pancreatic ductal carcinoma compared to nonmalignant pathological states. Clin Cancer Res 3: 993–998.

118.

Tan

Goedegebuure

Belt

Flaherty

Sankpal

Gillanders

. (2009) Disruption of CCR5-dependent homing of regulatory T cells inhibits tumor growth in a murine model of pancreatic cancer. J Immunol 182: 1746–1755.

119.

Thomas

Santarsiero

Lutz

Armstrong

Chen

Huang

. (2004) Mesothelin-specific CD8(+) T cell responses provide evidence of in vivo cross-priming by antigen-presenting cells in vaccinated pancreatic cancer patients. J Exp Med 200: 297–306.

120.

Tong

Papayoti

Netto

Armstrong

Ordonez

Lawson

. (2001) Growth-inhibitory effects of CD40 ligand (CD154) and its endogenous expression in human breast cancer. Clin Cancer Res 7: 691–703.

121.

Toubaji

Achtar

Provenzano

Herrin

Behrens

Hamilton

. (2008) Pilot study of mutant ras peptide-based vaccine as an adjuvant treatment in pancreatic and colorectal cancers. Cancer Immunol Immunother 57: 1413–1420.

122.

Van Elsas

Hurwitz

Allison

. (1999) Combination immunotherapy of B16 melanoma using anti-cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and granulocyte/macrophage colony-stimulating factor (GM-CSF)-producing vaccines induces rejection of subcutaneous and metastatic tumors accompanied by autoimmune depigmentation. J Exp Med 190: 355–366.

123.

Van Kooten

Banchereau

. (2000) CD40-CD40 ligand. J Leukoc Biol 67: 2–17.

124.

Vasef

Ross

Cohen

(1999) Telomerase activity in human solid tumors. diagnostic utility and clinical applications. Am J Clin Pathol 112(Suppl. 1): S68–S75.

125.

Von Hoff

Ervin

Arena

Chiorean

Infante

Moore

. (2013) Increased survival in pancreatic cancer with nab-paclitaxel plus gemcitabine. N Engl J Med 369: 1691–1703.

126.

Vonderheide

Flaherty

Khalil

Stumacher

Bajor

Hutnick

. (2007) Clinical activity and immune modulation in cancer patients treated with CP-870,893, a novel CD40 agonist monoclonal antibody. J Clin Oncol 25: 876–883.

127.

Wang-Gillam

Nywening

Sanford

Lockhart

Suresh

Tan

. (2015) Phase IB study of FOLFIRINOX plus PF-04136309 in patients with borderline resectable and locally advanced pancreatic adenocarcinoma (PC). ASCO Meeting Abstracts 33: 338.

128.

Wedén

Klemp

Gladhaug

Møller

Eriksen

Gaudernack

. (2011) Long-term follow-up of patients with resected pancreatic cancer following vaccination against mutant K-ras. Int J Cancer 128: 1120–1128.

129.

Weinstock

Mcdermott

(2015) Targeting PD-1/PD-L1 in the treatment of metastatic renal cell carcinoma. Ther Adv Urol 7: 365–377.

130.

Wilkie

Burbridge

Chiapero-Stanke

Pereira

Cleary

Van Der Stegen

. (2010) Selective expansion of chimeric antigen receptor-targeted T-cells with potent effector function using interleukin-4. J Biol Chem 285: 25538–25544.

131.

Wobser

Keikavoussi

Kunzmann

Weininger

Andersen

Becker

(2006) Complete remission of liver metastasis of pancreatic cancer under vaccination with a HLA-A2 restricted peptide derived from the universal tumor antigen survivin. Cancer Immunol Immunother 55: 1294–1298.

132.

Wolchok

Hoos

O’day

Weber

Hamid

Lebbé

. (2009) Guidelines for the evaluation of immune therapy activity in solid tumors: immune-related response criteria. Clin Cancer Res 15: 7412–7420.

133.

Yamamoto

Ueno

Kawaoka

Hazama

Fukui

Suehiro

. (2005) MUC1 peptide vaccination in patients with advanced pancreas or biliary tract cancer. Anticancer Res 25: 3575–3579.

134.

Yanagimoto

Takai

Satoi

Toyokawa

Takahashi

Terakawa

. (2005) Impaired function of circulating dendritic cells in patients with pancreatic cancer. Clin Immunol 114: 52–60.

135.

Zhao

Obermann

Von Wasielewski

Haile

Manns

Korangy

. (2009) Increase in frequency of myeloid-derived suppressor cells in mice with spontaneous pancreatic carcinoma. Immunology 128: 141–149.

136.

Zhao

Moon

Carpenito

Paulos

Liu

Brennan

. (2010) Multiple injections of electroporated autologous T cells expressing a chimeric antigen receptor mediate regression of human disseminated tumor. Cancer Res 70: 9053–9061.