Immunotherapy for Pancreatic Cancer

The morbidity rate of pancreatic cancer has increased globally over the last 50 years and it is the most difficult cancer to cure. However, treatments for pancreatic cancer are rapidly advancing. Combination chemotherapy using gemcitabine and nab-paclitaxel (GnP) or fluorouracil, leucovorin, irinotecan, and oxaliplatin (FOLFIRI-NOX) is the standard first-line therapy. However, small molecule agents may cause side-effects, such as myelosuppression. Immunotherapy for cancer has attracted attention and was recognized as the most important scientific advance in 2013 by the journal Science. Immunotherapies include effector cell therapy, treatment via immune checkpoint inhibitors, and cancer vaccines. Currently, only immune checkpoint inhibitors are used for treating pancreatic cancer. Pembrolizumab is an immune checkpoint inhibitor that can be used to treat pancreatic cancer and is indicated for microsatellite instability-high/DNA mismatch repair deficient (MSI-H/dMMR) solid tumors. MSI-H generates neoantigens, the expression of which increases the number of tumor-infiltrating lymphocytes. Through this mechanism, pembrolizumab can significantly improve the clinical condition of patients. However, MSI-H is present in only 2% of pancreatic cancers, such that 98 out of 100 patients receive no benefit from pembrolizumab. IL-13R α 2 is a cancer-testis antigen targeted by the immunotoxin, IL-13 ( Pseudomonas exotoxin). A total of 70% of pancreatic cancers strongly express IL-13R α 2, and IL-13PE shows anti-cancer effects in orthotopic mouse models. Clinical trials of IL-13PE, for the treatment of glioblastoma multiforme and adrenocortical carcinoma, are currently being performed. Immunotherapy will likely emerge as the fourth pillar of cancer treatment, along with surgery, chemotherapy, and radiation


Anti-cancer therapy for pancreatic cancer
Pancreatic cancer is one of the deadliest known cancers; the 5-year survival rate is less than 10%, which is the lowest among all solid cancers. Moreover, the morbidity of pancreatic cancer continues to increase globally. In Japan, the morbidity of pancreatic cancer increased about sixfold between 1975 and 2015. Pancreatic cancer was the fifth leading cause of cancer death among men and the third leading cause among women in 2017. For this reason, rapid development of therapies for pancreatic cancer has occurred. Chemotherapy using small molecule agents is the primary treat-ment for unresectable pancreatic cancer. The gemcitabine plus nab-paclitaxel (GnP) 1) and fluorouracil, leucovorin, irinotecan, and oxaliplatin (FOLFIRINOX) 2) regimens are the first-line therapies for pancreatic cancer (Figure-1). However, both regimens have side-effects that should be monitored, such as myelosuppression. In addition, FOLFIRINOX causes febrile neutropenia in more than 20% of patients. Numbness and edema of the hands and feet are also common, and are difficult to treat. When these side-effects become severe, discontinuation of therapy or switching to another therapy should be considered. Although rare, intestinal pneumonia can also occur, and can be difficult to treat. It is important that the chemotherapy treatment continues with consideration of the patientʼs physical condition and the potential side-effects. Current standard chemotherapy alone using small molecule agents cannot eradicate pancreatic cancer. Therefore, more powerful tools are required to treat this deadly disease.

Immunotherapy for pancreatic cancer
Immunotherapy for cancer was recognized as the most important scientific advance in 2013 by the journal Science, which is among the most influential scientific journals; widespread clinical application of immunotherapy is expected in the future 3) . Currently, surgery, chemotherapy, and radiotherapy are considered the three major pillars of pancreatic cancer treatment, and immunotherapy will likely emerge as the fourth pillar ( Figure-2). Immunotherapies include effector cell therapy, treatment via immune checkpoint inhibitors, and cancer vaccines (Figure-3). At this time, only immune checkpoint inhibitors are used for treating pancreatic cancer.

Immune checkpoint inhibitors for pancreatic cancer
The first immune checkpoint inhibitor to be approved by the US Food and Drug administration (US FDA) was ipilimumab, on March 25, 2011. Ipilimumab is a cytotoxic T-lymphocyte antigen 4 (CTLA-4)-blocking antibody indicated for the treatment of melanoma, where CTLA-4 is a negative regulator of T-cell activation. Ipilimumab binds to CTLA-4 and blocks interaction with its ligands, CD80 and CD86. Blockade of CTLA-4 results in augmented T-cell activation and proliferation, including of tumor-infiltrating T-cells. Ipilimumab is indicated for renal cell carcinoma and MSI-H/dMMR metastatic colorectal cancer, but not for pancreatic cancer. Pembrolizumab and nivolumab were the next immune checkpoint inhibitors to be approved by the US FDA, in 2014. These programmed death receptor-1 (PD-1) blocking antibodies are used to treat melanoma, Hodgkin lymphoma, and solid carcinomas. PD-1 controls the effector phase of T-cell attack by attenuating tumor antigen-specific signals. Pembrolizumab and nivolumab bind to the PD-1 receptor and block interaction with its ligands, PD-L1 and PD-L2, thus enhancing the immune response towards the cancer. In Japan, pembrolizumab is the only immune checkpoint inhibitor that can be used for treating pancreaticc cancer; it is indicated for MSI-H/dMMR solid tumors.
Mismatch repair genes, such as MLH-1, PMS-2, MSH-2 and MSH-6, play a role in DNA repair pathways, and loss of function of these genes results in dMMR tumors in association with changes in the size of microsatellites (i.e., MSI). MSI-induced frameshift mutations lead to the generation of a significant number of neoantigens, which increases the number of tumor-infiltrating lymphocytes. According to this mechanism, treatment with immune checkpoint inhibitors including ipilimumab, pembrolizumab, and nivolumab leads to significant clinical improvements in patients with MSI-H/ dMMR solid tumors.
Pembrolizumab should be used for the treatment of pancreatic cancer characterized by MSI-H/ dMMR tumors. However, the cancer with the highest rate of MSI-H/dMMR tumors is in fact endometrium cancer (17% vs.~2% for pancreatic cancer). Thus, 98 out of 100 patients with pancreatic cancer will not benefit from pembrolizumab, so the development of an immunotherapy that can be used in more patients with pancreatic cancer is important.
Side-effects have also been reported for pembrolizumab; for example, the immune system may be induced to attack not only cancer cells, but also normal organs and tissues in any area of the body. Pneumonitis, colitis, hepatitis, nephritis, impaired hormone gland functioning, and skin problems are frequently observed, and can be severe or even lifethreatening. Side-effects of chemotherapy involving small molecule immune checkpoint inhibitors should be monitored.

Other immunotherapies for cancer
Immunotherapies other than immune checkpoint inhibitors include effector cell therapy and cancer vaccines. The first effector cell therapy, sipuleucel-T, Recently, an important immunotherapeutic was approved in Japan, named tisagenlecleucel, which was approved by the US FDA on August 30, 2017. This chimeric antigen receptor T cell (CAR-T) therapy is used in patients with relapsed or refractory B-cell acute lymphoblastic leukemia (ALL), and in those with relapsed or refractory large B-cell lymphoma. CAR-T therapy is a novel approach to cancer treatment, in which a patientʼs T cells are genetically modified to express synthetic chimeric antigen receptors that redirect T cell specificity toward tumor-associated antigens. This therapy shows impressive efficacy in hematological malignancies. Therefore, it may have applications in solid tumors, including pancreatic cancer.

Interleukin-13 receptor alpha 2 as a target of cancer vaccines
An important type of cancer immunotherapy is cancer vaccines, including DNA and peptide vaccines, hapten-modified vaccines such as M-Vax, and immunotoxins. Our laboratory examines immunotoxin targeting IL-13 receptor α2 (IL-13Rα2). IL-13Rα2 is a high affinity receptor for the Th2derived cytokine IL-13. It is possible to create a nonsignaling decoy receptor for IL-13 because it does not exhibit canonical JAK-STAT signaling activity. However, some reports have shown that IL-13Rα2 can also signal through activator protein-1 4) 5) . IL-13Rα2 strongly expresses male germ cells and some types of cancers are coded on X chromosomes. It has the characteristics of a cancertestis antigen, which are groups of proteins with an important role in cancer development and immunotherapies. Cancer-testis antigens serve as a locus of immune activation and are often correlated with tumor progression. These antigens are human in origin and differ from neoantigens through genetic abnormalities. IL-13Rα2 is a suitable target for immunotherapy because it is expressed only in cancer, and not in normal tissues (excluding the testis) (Figure-4).

IL-13Rα2 promotes tumor invasion and metastasis in pancreatic cancer
In 2005, it was reported in the journal Nature that Juntendo Medical Journal 66 (3), 2020

Figure-4
Cancer-specific antigens targeted by cancer immunotherapy IL-13Rα2 promotes lung metastasis in breast cancer. That report indicated that IL-13Rα2 mediates the signaling pathway and is associated with cancer progression. Therefore, we explored the role of IL-13Rα2 in pancreatic cancer, and showed that it increases pancreatic cancer invasion and metastasis through the activation of extracellular signal-regulated kinase 1/2, activator protein-1 and nuclear factor nuclear factor kappa B 6) . IL-13Rα2-positive cancer showed greater invasiveness and metastasis than IL-13Rα2-negative cancer in an orthotopic mouse model.

Immunotoxin targeting IL-13Rα2
A recombinant chimeric fusion protein comprised of human IL-13 and a truncated form of Pseudomonas exotoxin (IL-13PE) has been produced (Figure-5) 7) 8) . The correctly folded IL-13PE, a type of immunotoxin, is highly cytotoxic to IL-13Roverexpressing tumor cells in many types of human carcinoma. IL-13PE induces apoptosis in tumor cells through both the classical pathway and mitochondrial cytochrome C release. On the other hand, IL-13PE is not cytotoxic to hemopoietic cells, such as monocytes, B cells, T cells, bone marrow cells, and nonhemopoietic normal cells, and does not mediate apoptosis of normal cells not expressing IL-13Rα2.

Pancreatic cancer therapy using IL-13PE
More than 90% of glioblastomas express IL-13Rα2, but it remains unclear how often IL-13Rα2 expression occurs in pancreatic cancer. Therefore, we assessed IL-13Rα2 expression in pancreatic cancer cells. Approximately 70% of pancreatic cancer cells moderately or strongly expressed IL-13Rα2. Using real-time imaging with green fluorescent protein (GFP), the anti-tumor effect of IL-13PE was examined in a mouse model of pancreatic cancer. GFP-labeled pancreatic cancer cells were orthotopically implanted and observed by irradiation ( Figure-6). IL13-PE significantly suppressed tumor growth and prolonged the mean survival time in an orthotopic pancreatic cancer mouse model. Similar results were observed in mice xenografted with a surgically resected human pancreatic tumor 9) .
However, about 30% of pancreatic cancer cases negative for IL-13Rα2 do not receive any benefit from treatment with IL-13PE, which showed antitumor effects only in IL-13Rα2-positive cancers. To address this issue, we developed a technique for upregulating the expression of IL-13Rα2. Initially, differences between IL-13Rα2-positive and -negative We showed that histone modification mostly affects the expression of IL-13Rα2 10) . Histone modification is an epigenetic modification that causes heritable changes in gene expression (active and inactive genes) that do not involve changes in the underlying DNA sequence. In pancreatic cancers showing IL-13Rα2 expression, all IL-13Rα2-positive cancer cells show increased acetylation at the promoter site of IL-13Rα2. Moreover, histone deacetylase (HDAC) inhibitors increased the expression of IL-13Rα2 in IL-13Rα2-negative cells, but not in IL-13Rα2positive cells. We then examined the efficacy of a combination therapy (HDAC inhibitors plus IL-13PE) for IL-13Rα2-negative pancreatic cancer. The HDAC inhibitors enhanced the anti-tumor effect of IL-13PE in IL-13Rα2-negative cancer cells in a mouse model of pancreatic cancer.

Clinical application of IL-13PE
IL-13PE has a strong anti-tumor effect on IL-13Rα2-positive cancer. Therefore, possible clinical applications of IL-13PE have been investigated.
Clinical trials have been performed regarding IL-13PE treatment of glioblastoma multiforme (GBM), adrenocortical carcinoma, and pediatric diffuse intrinsic pontine glioma. Previous phase IIII studies have explored treatments for GBM 11)-14) . In a phase III study, the anti-tumor effects of IL-13PE and Gliadel were limited; thus, the efficacy of a newly approved drug for GBM was examined, but it did not show significant superiority to IL-13PE. Clinical trials of adrenocortical carcinoma 15) and pediatric diffuse intrinsic pontine glioma 16) have also been performed. In a study on adrenocortical carcinoma, the effects of systemic intravenous administration of IL-13-PE were first assessed, mainly through locoregional administration 15) . The drug proved safe, but all tested patients developed high levels of neutralizing antibodies during treatment. Immunodepletion before IL-13-PE treatment should thus be considered.

Future treatments of pancreatic cancer
Both chemotherapy and immunotherapy for pancreatic cancer have been investigated. In the future, immunotherapy may become the first-line treatment for pancreatic cancer, and could reduce the need for surgical intervention.