Where Do We Stand with Immunotherapy for Advanced Pancreatic Ductal Adenocarcinoma: A Synopsis of Clinical Outcomes

Pancreatic cancer is the seventh leading cause of cancer-related mortality in both sexes across the globe. It is associated with extremely poor prognosis and remains a critical burden worldwide due to its low survival rates. Histologically, pancreatic ductal adenocarcinoma (PDAC) accounts for 80% of all pancreatic cancers; the majority of which are diagnosed at advanced stages, which makes them ineligible for curative surgery. Conventional chemotherapy provides a five-year overall survival rate of less than 8% forcing scientists and clinicians to search for better treatment strategies. Recent discoveries in cancer immunology have resulted in the incorporation of immunotherapeutic strategies for cancer treatment. Particularly, immune-checkpoint inhibitors, adoptive cell therapies and cancer vaccines have already shifted guidelines for some malignancies, although their efficacy in PDAC has yet to be elucidated. In this review, we summarize the existing clinical data on immunotherapy clinical outcomes in patients with advanced or metastatic PDAC.


Introduction
Pancreatic cancer is the seventh leading cause of cancer-related mortality in both sexes across the globe [1]. It remains a critical burden worldwide due to its low survival rates and extremely aggressive nature [2]. A total of 95% of pancreatic malignancies arise from exocrine parts (ductal epithelium, acinar cells and connective tissue), and another 5% develop from endocrine parenchyma [3]. Histologically, pancreatic ductal adenocarcinoma (PDAC) accounts for 80% of all pancreatic cancers [4], and the majority of cases are diagnosed at advanced stages ( Figure 1) [5]. Localized cases can be treated with surgery, however, the five-year overall survival (OS) rate does not exceed 25% [6]. Unfortunately, there are no curative strategies for advanced stages, and palliative chemoradiotherapy reaches a five-year OS rate of less than 8% [7]. Chemotherapeutic choices of treatment include either modified FOLFIRINOX (5-fluorouracil, leucovorin, irinotecan, oxaliplatin) [8], gemcitabine monotherapy or gemcitabine in combination with nab-paclitaxel [9].
Recent discoveries in cancer immunology resulted in the incorporation of immunotherapeutic strategies for the treatment of various solid and hematologic malignancies [10].
Multiple clinical trials showed the higher efficacy of immune-checkpoint inhibitors (ICIs) for melanoma, lung cancer, renal cell carcinoma, colorectal cancer and hepatocellular carcinoma [11][12][13][14][15]. Adoptive chimeric antigen receptor (CAR) T-cell therapy is another immunotherapeutic tool being established as effective for hematologic malignancies, particularly relapsed/refractory B cell lymphoma [16] or mantle-cell lymphoma [17]. Unfortunately, early-phase clinical trials have shown limited responses to immunotherapy hepatocellular carcinoma [11][12][13][14][15]. Adoptive chimeric antigen receptor (CAR) T-cell therapy is another immunotherapeutic tool being established as effective for hematologic malignancies, particularly relapsed/refractory B cell lymphoma [16] or mantle-cell lymphoma [17]. Unfortunately, early-phase clinical trials have shown limited responses to immunotherapy in patients with PDAC [18,19]. Nevertheless, immunotherapy is still deemed the most likely way to improve prognosis for patients with advanced PDAC (aPDAC) in the near future [20]. In this review, we provide an overview of the clinical outcomes of immunotherapy in patients with advanced and/or metastatic PDAC.  [21]. (B) Morphological differences between normal (left) and malignant (right) pancreatic tissue. (C) TNM classification of PDAC.

Immune-Checkpoint Inhibitors
ICIs have heralded a new era in clinical oncology [22]. ICIs are monoclonal antibodies that target immune checkpoints (e.g., cytotoxic T-lymphocyte antigen-associated protein
To sum up, the existing results do not show encouraging outcomes for CTLA-4 inhibitors in patients with aPDAC. Strikingly, even the combination of the PD-L1 inhibitor durvalumab + CTLA-4 inhibitor tremelimumab is not significantly better than SoC chemotherapy. This data emphasizes the need for further studies on novel combinatorial approaches able to overcome the unique immunosuppressive and fibrotic morphological features of PDAC that potentially play a major role in the inhibition of existing ICI-based regimens. Moreover, further trials should be designed for the head-to-head comparison of a single ICI or a combination of CTLA-4 + PD-1 inhibitors to the current SoC or SoC + ICI.

Blockade of Programmed Cell Death-1 with Its Ligands
Pembrolizumab (PEMBRO) and nivolumab (NIVO) are humanized monoclonal antibodies (mAbs) inhibiting the PD-1 inhibitory checkpoint [34]. They have been granted FDA approval for the treatment of melanoma, lung cancer and aPDAC with MSI-H status [19].
A single-arm phase 2 trial (NCT01876511) reported that cohort C (non-colorectal cancer patients [n = 7 out of whom n = 4 with PDAC] with a mismatch repair deficiency [dMMR] or a high microsatellite instability [MSI-H]) treated with PEMBRO as a ≥2 line of therapy reached an ORR of 71% with a median PFS of 5.4 months [35]. The authors concluded that PEMBRO alone showed a durable clinical efficacy in the MSI-H patients from cohort C [36]. Later reports from the NCT01876511 trial also observed durable response in the cohort of patients (n = 86) with PDAC (ORR 53% [95% CI: 42% to 64%]; 21% of patients reached complete response [CR]) [37]. A recent report from the multicenter KEYNOTE-158 trial reported that out of n = 22 patients only n = 4 (18.2%) had either CR or a partial response [PR] [38]. Nonetheless, the prevalence of PDAC patients with MSI-H is between 0.8% and 2% [36], which stresses the extreme necessity for searching for other therapeutic strategies effective for the majority of PDAC patients.
Finally, a randomized phase 2 CheckPAC study (NCT02866383) established that NIVO in combination with IPI and stereotactic body radiotherapy (SBRT) of 15 Gy reached a 37.2% ORR in patients with metastatic PDAC [42]. The authors concluded that the studied regimen showed a favorable efficacy, and further studies are currently ongoing. Nevertheless, the role of SBRT is unknown.
In summary, the current evidence supports that PEMBRO is effective for aPDAC patients with MSI-H status, although the data were obtained from basket single-arm trials primarily powered by elucidating PEMBRO's efficacy in colorectal cancer patients. NIVO in combination with (1) chemotherapy or (2) IPI + SBRT showed meaningful response rates; however, the head-to-head comparison with current SoC chemotherapy regimens is necessary to precisely elucidate the safety and efficacy profiles of this regimen.
A dose-escalating phase 1b trial (NCT02323191) determined the role of the PD-L1 inhibitor Atezolizumab (Atezo) in combination with Emactuzumab, an inhibitor of the colony-stimulating factor-1 receptor (CSF1R) in patients with solid tumors [43]. Gomez-Roca et al. reported that the ORRs in treatment-naïve aPDAC patients reached 9.8% and 12.5% for patients previously treated with other ICI-based regimens, albeit a separate analysis of patients with only aPDAC was not conducted [43]. Further clinical studies were not warranted due to the full potential of CSF1R being unknown and the high rate of grade 3-4 irAEs.
Finally, despite ICIs having demonstrated their superior efficacy in aPDAC with MSI-H status, most phase 1 and 2 clinical trials failed to demonstrate any superior clinical efficacy in a majority of aPDAC patients as compared to the current SoC. Perhaps further data generated from ongoing clinical trials will reveal encouraging results for a combination of ICIs + radiotherapy and/or chemotherapy (Table 1). Nonetheless, further studies are critically needed to determine the mechanisms of PDAC's resistance to ICI therapy. Rand-randomization; NIVO-nivolumab; IPI-ipilimumab; PEMBRO-pembrolizumab; ATEZO-atezolizumab; TRAEs-treatment-related adverse events; DLTs-dose-limiting toxicities; PFS-progression-free survival; ORR-objective response rate; mAb-monoclonal antibody; CTLA-4-cytotoxic T-lymphocyte-associated antigen 4; PD-1-programmed cell death protein 1; SCCHN-squamous cell carcinoma of head and neck; RCC-renal cell carcinoma; DOR-duration of response; TTR-time to response; EGFR-epidermal growth factor receptor; TGFβ-transforming growth factor β.

Adoptive CAR T-Cell Therapy
Adoptive CAR (chimeric antigen receptor) T-cell therapy is a technology of the ex vivo expansion of a patient's own T cells that have been genetically engineered to express CAR Biomedicines 2022, 10, 3196 7 of 13 that recognizes a particular tumor antigen [45]. To date, no CAR T-cell therapy has been approved for the treatment of solid malignancies [45]. Regarding PDAC, a few clinical trials have demonstrated limited efficacy.
A single-arm phase 1 study (NCT01869166) determined the safety and efficacy of anti-EGFR (epidermal growth factor receptor) CAR T cells [46]. Among n = 14 pre-treated metastatic PDAC patients, the ORR reached 28.6% with a median PFS and OS of 3 and 4.9 months, respectively [46]. Each patient experienced a treatment-related adverse event (TRAE) of different severity [46]. Further trials were not supported due to the limited efficacy and the fact that EGFR is expressed by a wide range of tissues, which may explain the high incidence of TRAEs.
Another single-arm phase 1 basket trial (NCT01935843) elucidated the safety and efficacy of anti-HER-2 (human epidermal growth factor receptor 2). The median PFS reached 4.8 months in n = 11 aPDAC patients with more than 50% of HER-2 positive tumor cells. n = 1 (9%) reached PR, and n = 5 (45%) achieved stable disease (SD) [47]. No instances of high-grade lymphocytopenia or cytokine release syndrome were detected, and most low-grade toxicities were reversible [47]. The therapy showed encouraging clinical activity, and further trials are ongoing.
Based on promising preclinical mouse studies, two trials elucidated the clinical outcomes of anti-mesothelin CAR T cell therapy. A phase 1 trial (NCT01897415) showed that among n = 6 patients with aPDAC, none experienced severe TRAEs [48]. n = 2 (33.3%) reached SD [48]. Another phase 1 basket trial (NCT02159716) showed encouraging safety profiles in patients with chemo-refractory solid tumors [49]. n = 11 (73.3%) of patients achieved SD, and additional larger trials are currently ongoing.
To date, knowledge regarding the clinical efficacy of CAR therapy in patients with aP-DAC is very limited, although it remains a viable and promising topic of pancreatic cancer research. The identification of an ideal target is a major challenge for adoptive cell therapy in PDAC. Secondly, PDAC's immune microenvironment comprised of macrophages, cancer-associated fibroblasts, myeloid-derived suppressor cells, dendritic cells, B cells and infiltrating regulatory T cells [51]. These cells can create a physical barrier for trafficking T cells and suppress T cell activation, resulting in a diminished efficacy of CAR T-cell therapy [52]. Overcoming those hurdles may one day result in successful implications of adoptive cell therapies for advanced and/or metastatic PDAC.

Cancer Vaccines
Cancer vaccines represent another promising strategy for PDAC treatment [53]. Vaccines can boost anti-tumor immunity by transferring the tumor antigens (Figure 3) in the form of cells, proteins or nucleic acids [53]. A number of clinical trials have elucidated the clinical efficacy of vaccines in patients with severely progressed PDAC.

Cell-Based Vaccines
A phase 2 open-label randomized trial (NCT02243371) determined the efficacy of the GVAX and CRS-207 vaccines with or without NIVO in patients with metastatic PDAC that progressed on one prior chemotherapy regimen [54]. GVAX is a cell-based vaccine that transfers an allogeneic tumor cell, engineered to express granulocyte-macrophage colonystimulating factor (GM-CSF) [55]. CRS-207 is a microorganism-based vaccine that transfers a live-attenuated Listeria monocytogenes (Lm) engineered to express the PDAC-associated antigen mesothelin [56]. Recruited patients received Cyclophosphamide + GVAX + CSR-207 with (cohort A, n = 51) or without (cohort B, n = 42) NIVO [54]. The median OS reached 5.9 (95% CI: 4.7 to 8.6) and 6.1 (95% CI: 3.5 to 7) months in cohorts A and B, respectively [54]. The ORR was 4% and 2% in cohorts A and B, respectively [54]. The trial failed to meet its primary endpoint of OS improvement, and durable response rates were not registered.

Cell-Based Vaccines
A phase 2 open-label randomized trial (NCT02243371) determined the efficacy of the GVAX and CRS-207 vaccines with or without NIVO in patients with metastatic PDAC that progressed on one prior chemotherapy regimen [54]. GVAX is a cell-based vaccine that transfers an allogeneic tumor cell, engineered to express granulocyte-macrophage colony-stimulating factor (GM-CSF) [55]. CRS-207 is a microorganism-based vaccine that transfers a live-attenuated Listeria monocytogenes (Lm) engineered to express the PDACassociated antigen mesothelin [56]. Recruited patients received Cyclophosphamide + GVAX + CSR-207 with (cohort A, n = 51) or without (cohort B, n = 42) NIVO [54]. The median OS reached 5.9 (95% CI: 4.7 to 8.6) and 6.1 (95% CI: 3.5 to 7) months in cohorts A and B, respectively [54]. The ORR was 4% and 2% in cohorts A and B, respectively [54]. The trial failed to meet its primary endpoint of OS improvement, and durable response rates were not registered.
Another phase 2 (NCT02004262) randomized trial (ECLIPSE study) compared the efficacy of Cyclophosphamide + GVAX + CRS-207 (arm A) and CRS-207 (arm B) to a physician's choice of an SoC chemotherapy [57] in patients with metastatic PDAC who previously failed on >2 lines of chemotherapy. The median OS reached 3.7 (95% CI: 2.9 to 5.3), 5.4 (95% CI: 4.2 to 6.4) and 4.6 (95% CI: 4.2 to 5.7) months in arms A, B and C, respectively [57]. No significant difference as compared to control arm C was registered. The authors concluded that the combination of GVAX + CRS-207 failed to show higher efficacy as compared to SoC chemotherapy in patients with metastatic PDAC [57].
A phase 3 randomized PILLAR trial (NCT01836432) determined the clinical role of Algenpantucel-L + FOLFIRINOX (arm A, n = 145), compared to FOLFIRINOX (arm B, n = Another phase 2 (NCT02004262) randomized trial (ECLIPSE study) compared the efficacy of Cyclophosphamide + GVAX + CRS-207 (arm A) and CRS-207 (arm B) to a physician's choice of an SoC chemotherapy [57] in patients with metastatic PDAC who previously failed on >2 lines of chemotherapy. The median OS reached 3.7 (95% CI: 2.9 to 5.3), 5.4 (95% CI: 4.2 to 6.4) and 4.6 (95% CI: 4.2 to 5.7) months in arms A, B and C, respectively [57]. No significant difference as compared to control arm C was registered. The authors concluded that the combination of GVAX + CRS-207 failed to show higher efficacy as compared to SoC chemotherapy in patients with metastatic PDAC [57].
Dendritic cells (DCs) are antigen-presenting cells and play a crucial role in the antitumor immune response. A phase 1 single-arm trial (NCT01410968) determined the role of DCs isolated from the peripheral blood of PDAC patients with HLA-A2 positive status. DCs were pulsed with three distinct A2-restricted peptides: (1) human telomerase reverse transcriptase (hTERT, TERT572Y); (2) carcinoembryonic antigen (CEA, Cap1-6D) and (3) surviving (SRV.A2) [60]. The median OS reached 7.7 months, and n = 4 (33.3 %) reached SD [60]. The treatment was well tolerated, and the flow cytometry analysis revealed that patients with SD had a high expansion of antigen-specific T cells [60]. This method was considered promising because DCs can be pulsed with other synthetic peptides, and further larger trials were recommended.

Peptide-Based Vaccines
The vaccine KIF20A-66 is a human leukocyte antigen (HLA)-A24-restricted cytotoxic T cell epitope derived from KIF20A, a member of the kinesin superfamily protein 20A that is markedly upregulated in PDAC [61]. A phase 1/2 single-arm trial (UMIN000004919) reported that vaccinated gemcitabine-pre-treated patients with metastatic PDAC had a median OS and PFS of 4.7 and 1.9 months, respectively [62]. n = 21 (72%) of patients reached SD [62]. Asahara et al. concluded that KIF20A-66 showed higher survival rates in patients with metastatic PDAC as compared to the best supportive care, and further trials were encouraged.
A phase 2 single-arm non-randomized VENUS-PC trial (UMIN000008082) determined the role of HLA-A*2402-restricted KIF20A-derived peptide vaccine in combination with the gemcitabine and antiangiogenic vaccines targeting vascular endothelial growth factor receptor 1 and 2 (VEGFR1, 2) [63]. The median OS reached 9 months with an ORR of 10.8% in n = 38 patients who had at least one allele of HLA-A*2402 (matched).
Finally, commonly tested KRAS and telomerase (GV1001)-targeting vaccines failed to show durable responses and/or superiority to gemcitabine in phase 2/3 clinical trials [64]. A phase 3 randomized TeloVac trial showed no significant difference in the OS for treatment-naïve patients with metastatic PDAC treated with chemotherapy or the GV1001 +/− chemotherapy vaccine [65].
Most of the trials determining the role of vaccines in patients with advanced PDAC failed to show durable response. Perhaps a critical factor responsible for such failure is the tumor microenvironment, which is characterized by an abundance of mesenchymal origin fibroblasts, blood vessels and tumor-infiltrating immune cells surrounded by extracellular matrix [66]. Those factors can inhibit the immune response, thus facilitating cancer escape from immunosurveillance [66]. In addition, vaccine therapy is challenged by a complex process of vaccine synthesis and the absence of a validated method to identify and/or measure the immune response to the vaccine. Nevertheless, further assessment in larger trials is necessary, especially in combination with other therapeutic strategies ( Table 2). Table 2.

Conclusions
PDAC is associated with an extremely poor survival rate and prognosis if diagnosed at late stages. To date, immunotherapy represents the biggest hope for improving clinical outcomes for patients with advanced/metastatic PDAC. Although PEMBRO has been approved for the treatment of aPDAC patients with MSI-H status, PDAC has demonstrated remarkable resistance to immunotherapy in the majority of cases. Further trials are extremely necessary to determine the role of combination approaches utilizing various immunotherapeutic strategies. Importantly, further trials should focus on overcoming therapeutic resistance by targeting multiple immune defects with several immunotherapeutic arms. Early trials have already reported the synergistic effect of ICIs or CAR therapies with chemoradiotherapy, albeit safety profiles should be closely monitored. In addition, future studies should prioritize integrated or convergent targets that can reprogram the tumor microenvironment rather than focusing on the depletion of a single/particular target. Another important point to consider is that an immunotherapeutic strategy should be based on an individual characterization of the tumor microenvironment of each patient. Such approach can be achieved by deep profiling of the tumor tissue during the pre-treatment stage with high-throughput technologies. This will promote the personalization of therapy, thus increasing clinical outcomes in patients with advanced and metastatic PDAC. Overall, such integration may facilitate the establishment of effective therapeutic strategies for the majority of PDAC patients in the near future.