Immunotherapy For Pancreatic Cancer Research Articles

Abstract B076: Novel use of Listeria and gemcitabine to improve immunotherapy for pancreatic cancer

Abstract Introduction: Pancreatic ductal adenocarcinoma (pancreatic cancer), is the 4th leading cause of cancer deaths. This “silent killer” is characterized by its development of metastases often before the primary tumor can be detected, resulting in a five-year survival rate of only 4%. Gemcitabine and erlotinib, FDA-approved drugs for pancreatic cancer, improve median survival by less than six months in advanced stage patients, underscoring the need for additional and/or new alternative approaches. Cancer immunotherapy could be such new approach because of its great promise for metastatic cancer, however hurdles need to be overcome. The first hurdle is that tumor-associated antigens (TAA) used in most cancer vaccines are poorly immunogenic, and the second hurdle is immune suppression, particularly by myeloid-derived suppressor cells (MDSC) that prevent T cell activation in the tumor microenvironment (TME). To address these problems we have developed a novel strategy using an attenuated bacterium Listeria monocytogenes that delivers highly immunogenic antigens selectively into tumor cells of metastases and tumors combined with Gemcitabine (GEM) to reduce immune suppression through MDSC. We have cloned highly immunogenic recall antigens (RA) such as tetanus toxoid (TT), poliovirus (PV), and measlevirus (MV) antigens into the Listeria to activate existing memory T cells to foreign highly immunogenic antigens, which most individuals have been exposed to during childhood through vaccinations, and to deliver these antigens selectively into tumor cells. Memory T cells generated to the RA following childhood vaccinations circulate in their blood for life and can upon reactivation kill the infected tumor cells with high efficiency. We have previously demonstrated this with Listeria-TT in a triple-negative breast cancer model 4T1. Listeria and GEM also kill tumor cells through reactive oxygen species (ROS). In the current study we analyzed whether GEM could improve the efficacy of Listeria-TT immunotherapy in mice with pancreatic cancer (Panc-02 model). Experimental procedures: Tumor-bearing mice (pre-immunized with the human TT vaccine) were treated with GEM and Listeria-TT, and effects were analyzed on metastases, tumors, and MDCS. Results: We found that Listeria-TT combined with GEM resulted in a near complete elimination of metastatic pancreatic cancer (metastases and tumors), which was not possible with Listeria-TT or GEM alone. We also found that GEM reduced the MDSC population by 50% in the blood. Currently, we are analyzing CD8 T cell responses of treated and control mice with pancreatic cancer to immunodominant epitopes within the TT protein (TT1162-1169:SNWYFNHL and TT12-86-1294:LNIYYRRL) matching the H2-b haplotype. In conclusion, the combination of GEM and Listeria-TT eliminated metastatic pancreatic cancer in the Panc-02 model. We expect that this dramatic effect is a combination of immune-based and non-immune-based mechanisms. Future prospects: Since the cloned recall antigen fragments also contain many T cell epitopes recognized in humans, we expect that these constructs are directly translatable to human clinical trials in the near future. Finally, cancer patients could be tested in advance by revaccination with human childhood vaccines to determine to which antigens they react to best before starting the Listeria-recall antigen vaccine therapy. Based on patient's best response (most likely determined by MHC haplotype and the TME), a personalized therapy with the best combination of recall antigen(s) could be designed. Citation Format: Dinesh Chandra, Wilber Quispe-Tintaya, Arthee Jahangir, Alice Kwon, Rodrigo Alves da Silva, Lukman Solola, Ziqiang Yuan, Steven K. Libutti, Claudia Gravekamp. Novel use of Listeria and gemcitabine to improve immunotherapy for pancreatic cancer. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B076.

Commentary on from Bench to Bedside: A Comprehensive Review of Pancreatic Cancer Immunotherapy. Slow Improvements in Meaningful Clinical Benefit

Commentary on from Bench to Bedside: A Comprehensive Review of Pancreatic Cancer Immunotherapy. Slow Improvements in Meaningful Clinical Benefit

Current progress in immunotherapy for pancreatic cancer

Pancreatic cancer remains one of the most lethal cancers with few treatment options. Immune-based strategies to treat pancreatic cancer, such as immune checkpoint inhibitors, therapeutic vaccines, and combination immunotherapies, are showing promise where other approaches have failed. Immune checkpoint inhibitors, including anti-CTLA4, anti-PD-1, and anti-PD-L1 antibodies, are effective as single agents in immune sensitive cancers like melanoma, but lack efficacy in immune insensitive cancers including pancreatic cancer. However, these inhibitors are showing clinical activity, even in traditionally non-immunogenic cancers, when combined with other interventions, including chemotherapy, radiation therapy, and therapeutic vaccines. Therapeutic vaccines given together with immune modulating agents are of particular interest because vaccines are the most efficient way to induce effective anti-tumor T cell responses, which is required for immunotherapies to be effective. In pancreatic cancer, early studies suggest that vaccines can induce T cells that have the potential to recognize and kill pancreatic cancer cells, but the tumor microenvironment inhibits effective T cell trafficking and function. While progress has been made in the development of immunotherapies for pancreatic cancer over the last several years, additional trials are needed to better understand the signals within the tumor microenvironment that are formidable barriers to T cell infiltration and function. Additionally, as more pancreatic specific antigens are identified, immunotherapies will continue to be refined to provide the most significant clinical benefit.

MDSC-decreasing chemotherapy increases the efficacy of cytokine-induced killer cell immunotherapy in metastatic renal cell carcinoma and pancreatic cancer.

Adoptive immunotherapy using cytokine-induced killer (CIK) cells is a promising cancer treatment, but its efficacy is restricted by various factors, including the accumulation of myeloid-derived suppressor cells (MDSCs). In this study, we determine whether chemotherapeutic drugs that reduce MDSC levels enhance the efficacy of CIK cell therapy in the treatment of solid tumors. Fifty-three patients were included in this study; 17 were diagnosed with metastatic renal cell carcinoma (MRCC), 10 with advanced pancreatic cancer (PC), and 26 with metastatic melanoma (MM). These patients were divided into two groups: CIK cell therapy alone and CIK cell therapy combined with chemotherapy. Combining CIK cell therapy and chemotherapy increased 1-year survival rates and median survival times in MRCC and PC patients, but not in MM patients. The disease control rate did not differ between treatment groups for MRCC or MM patients, but was higher in PC patients receiving combined treatment than CIK cell treatment alone. These data suggest that addition of MDSC-decreasing chemotherapy to CIK cell therapy improves survival in MRCC and PC patients.

The current status and perspectives of immunotherapy in pancreatic adenocarcinomas

Pancreatic adenocarcinoma is one of the most highly malignant digestive system cancers, and current therapeutic strategies are often unsatisfactory. It is associated with a high mortality rate because of low resection rate, early metastasis, and poor chemoradiotherapy response. Identification and development of more efficacious therapies is urgently needed. The basic research promoted the development of immunotherapy for pancreatic cancer, and immunotherapy offered encouraging results in some preclinical trials during the last decade. The aim of this review is to summarize the recent advances in immunotherapy and to evaluate the future perspectives of immunotherapy in the treatment of pancreatic adenocarcinoma. Key words: Pancreatic neoplasms/TH; Immunotherapy; Review

Abstract B197: Focal adhesion kinase inhibition enables efficacy of checkpoint immunotherapy in pancreatic cancer

Abstract Checkpoint immunotherapeutics are promising agents with potential to improve patient outcomes. Unfortunately, to date, single agent immunotherapy has achieved limited clinical benefit in patients with pancreatic ductal adenocarcinoma (PDAC). This may be due to the presence of the uniquely immunosuppressive tumor microenvironment present in PDACs that creates a barrier to immune surveillance by T cells. Critical obstacles to immune surveillance in PDAC tumors include a dense desmoplastic stroma that acts as a barrier to T cell infiltration and high numbers of tumor-associated immunosuppressive cells, such as tumor-associates macrophages, MDSCs, and regulatory T cells (Tregs). To understand which signaling pathways in pancreatic tumor cells might drive this suppressive tumor microenvironment, we analyzed the correlation between hyper-activated signaling molecules and tumor infiltrating leukocytes using 50 human PDAC tumor tissues. Of the pathways evaluated, we found that focal adhesion kinase (FAK) activity is elevated in human PDAC which correlates with highly fibrotic tumors with poor CD8+ T cell infiltration. The oral FAK kinase inhibitor VS-4718, currently in phase 1 clinical evaluation, was tested to determine if it could overcome the immunosuppressive tumor microenvironment of PDAC. Single agent VS-4718 dramatically limited tumor progression resulting in a doubling of survival in the p48-CRE/KrasG12D/p53flox/+ PDAC mouse model (KPC mice). This alteration in tumor progression was associated with dramatically reduced tumor fibrosis, decreased numbers of Tregs and tumor-infiltrating myeloid cells, and anti-tumor polarization of tumor-associated macrophages. We postulated that the desirable effects of FAK inhibition on the tumor microenvironment might render PDAC tumors more sensitive to immunotherapy. Accordingly, we found that VS-4718 significantly potentiated the efficacy of anti-PD-1 and anti-CTLA4 antibodies in KPC mouse models with a near tripling of survival. We next assessed the mechanism of this potentiation. We found that FAK in tumor cells regulated pro-inflammatory and pro-fibrotic cytokine secretion. Furthermore, we found that shRNA knockdown of FAK in PDAC cells resulted in failure of PDAC cells to induce pro-fibrotic programs in fibroblasts. Importantly, the FAK inhibitor VS-4718 and FAK shRNA in the tumor cells were each effective in increasing CD8+ cytotoxic T cell infiltration into the PDAC tumors in vivo. These data suggest that FAK inhibition increases immune surveillance programs in PDAC tumors by overcoming the fibrotic and inflammatory microenvironment rendering tumors more responsive to immunotherapy. These data provide rationale for clinical evaluation of a FAK inhibitor in combination with a PD-1 or PD-L1 antibody in patients with pancreatic and other cancers. Citation Format: Hong Jiang, Brett L. Knolhoff, Samarth Hedge, Yu Zhu, John Herndon, Irina M. Shapiro, David T. Weaver, Jonathan A. Pachter, David G. Denardo. Focal adhesion kinase inhibition enables efficacy of checkpoint immunotherapy in pancreatic cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B197.

The Road to Stroma-Directed and Immunotherapy in Pancreatic Cancer

The Road to Stroma-Directed and Immunotherapy in Pancreatic Cancer

Abstract 3187: Depleting cells expressing fibroblast activation protein disrupts tumor-promoting desmoplasia

Abstract Solid tumors recruit and activate a heterogeneous population of non-transformed stromal cells (CASCs) in the tumor microenvironment (TME). There has been growing interest in the field to develop strategies to treat cancer and circumvent resistance to therapy by targeting CASCs. Defining the phenotypic subsets and the function of distinct subpopulations including myofibroblasts (defined by the expression of SMA) and the overlapping but distinct population of CASCs expressing Fibroblast Activation Protein (FAP) will be critical to informing the rational design of stromal-targeted therapies. FAP is a membrane serine protease that is selectively up-regulated in CASCs including cancer-associated fibroblasts (CAFs), mesenchymal stem cells (MSCs), and a subset of M2 macrophages, of virtually all common human epithelial cancers. Based on its selective expression on CASCs across a wide range of tumor types, we engineered FAP-specific chimeric antigen receptor expressing T cells (FAP-CAR T cells) and adoptively transferred them into tumor bearing mice to: 1) define the requirement for FAP+ cells for the accumulation and maintenance of a desmoplastic matrix; 2) define the relationship between the degree of desmoplasia, the capacity of FAP-CAR T cells to disrupt the tumor stroma and to inhibit tumor growth; and 3) determine whether FAP-CAR T cells can inhibit tumor growth through immune independent mechanisms. We found that FAP+ CACSs are responsible for maintaining the desmoplastic response and that conditional depletion following treatment with FAP-CAR T cells inhibits growth of human lung cancer xenografts and mouse pancreatic tumors in an immune-independent fashion. Taken together with recently published studies (1-3), our data provide important distinctions between the function of FAP+ CASCs and SMA+ myofibroblasts and provide support for further development of FAP+ stromal cell-targeted therapies for a wide range of solid tumors. Reference: 1. Feig, C., Jones, J. O., Kraman, M., Wells, R. J., Deonarine, A., Chan, D. S., et al. (2013). Targeting CXCL12 from FAP-expressing carcinoma-associated fibroblasts synergizes with anti-PD-L1 immunotherapy in pancreatic cancer. Proc Natl Acad Sci USA 110, 20212-20217. 2. Ozdemir, B. C., Pentcheva-Hoang, T., Carstens, J. L., Zheng, X., Wu, C. C., Simpson, T. R., et al. (2014). Depletion of carcinoma-associated fibroblasts and fibrosis induces immunosuppression and accelerates pancreas cancer with reduced survival. Cancer Cell 25, 719-734. 3. Wang, L. C., Lo, A., Scholler, J., Sun, J., Majumdar, R. S., Kapoor, V., et al. (2014). Targeting fibroblast activation protein in tumor stroma with chimeric antigen receptor T cells can inhibit tumor growth and augment host immunity without severe toxicity. Cancer Immunol Res 2, 154-166. Citation Format: Albert Lo, Liang-Chuan S Wang, John Scholler, James Monslow, Diana Avery, Rebecca A. Evans, David J. Bajor, Amy C. Durham, Elizabeth L. Buza, Robert H. Vonderheide, Carl H. June, Steven M. Albelda, Ellen Puré. Depleting cells expressing fibroblast activation protein disrupts tumor-promoting desmoplasia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3187. doi:10.1158/1538-7445.AM2015-3187

Abstract PL04-03: Fatal Attraction: A new story featuring the immune system and pancreatic cancer

Abstract Until recently, barriers to immunotherapy clinical successes for pancreatic cancer have been due to a lack of understanding of the immune pathways within the pancreatic tumor microenvironment that hinder successful immune responses. Recent advances have identified new targets for developing cancer specific vaccines and new targets for modulating the tumor microenvironment to allow more potent activation and improved access of vaccine induced immune responses. We are just learning that non-neoplastic cells, including cancer-associated myofibroblasts, regulatory T cells, dendritic cells, myeloid-derived suppressor cells, and tumor-associated macrophages, are hijacked by pre-invasive and invasive cancer cells to create a tolerogenic tumor microenvironment. Current research is focused on elucidating the mechanisms of this tolerogenic polarization within the tumor microenvironment with the goal of tipping the balance in favor of an anticancer immune response. With the recent advances in molecular technologies and the development of relevant pancreatic cancer mouse models, it is now within our reach to dissect the inhibitory pathways within the pancreatic tumor microenvironment. This will in turn, lead to new therapeutic opportunities that will eliminate these barriers and convert this deadly cancer into a treatable disease. Building on our extensive experience developing immunotherapies for pancreatic cancer, we are now developing combinatorial immunotherapies that combine T cell inducing agents with agents that modulate T cell down regulatory signals within the tumor microenvironment. These studies have shown for the first time that traditionally “non-immunogenic” cancers that do not respond to single agent immune modulating agents, can be converted into tumors susceptible to these agents if a T cell inducing vaccine is given together with these immune modulating agents. Results from both pre-clinical and clinical studies will be discussed.

Abstract A61: Exploring the PDAC-subtype-associated microenvironment in PDX models and patients

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease with dismal prognosis (1, 2). Despite extensive research and the discovery of several promising drug candidates, little progress in PDAC treatment has been reported in the last years (3, 4). Two facts can be behind these disappointing results. In one hand, although PDAC is still clinically treated as a single disease, three distinct molecular subtypes (classical, quasi-mesenchymal and exocrine) were recently described based on expression profiling of microdissected epithelial tumor cells of PDAC samples (5). Interestingly, in vitro studies revealed differences in drug response of two of the proposed subtypes for which cell lines were available. These results highlight the importance of considering PDAC as a heterogeneous disease and point to the stratification of patients as a possible way to improve PDAC treatment response. On another hand, an additional reason behind the limited efficacy of PDAC treatment might be the tumor microenvironment. PDAC is the solid tumor with the highest stromal content, which can account for up to 90% of the total tumor mass. The PDAC microenvironment is known to actively affect tumorigenesis (6) and to impair drug delivery (7). Thus, rendering the PDAC microenvironment as an appealing therapeutic target to improve PDAC care (8). We have developed a novel workflow to efficiently generate patient-derived orthotopic xenografts (PDX) and serum-free cell cultures from primary resected PDAC tumors. The established primary cell lines comprise for first time all three described PDAC subtypes. Additionally, when re-injected into immunodeficient mice, these cells generate xenografts with high pathological similarity to the original patient tumor, including a prominent stromal presence. To explore the differences in the microenvironment associated to the individual PDAC subtypes we have now generated gene expression profiles for the stroma of a number of xenografts from our PDX model representing all three subtypes. Besides, RNA sequencing from different sub-populations isolated from fresh primary human PDAC tumors (as separated by fluorescent activated cell sorting according to surface markers) may reveal interesting interactions between the different tumor compartments. We have developed a set of immunohistochemical markers to identify the PDAC-subtypes that can be used in patient paraffin sections. Hence, the RNAseq data of the different tumor populations can be also easily studied in the context of the different subtypes. We believe that these approaches will shed some light on how different stromal expression patterns are interconnected with different epithelial expression profiles and vice versa, and how this information can be ultimately exploited for patient stratification and therapy. 1. Hidalgo M. Pancreatic cancer. N Engl J Med 2010 2. Vincent A et al. Pancreatic cancer. Lancet, 2011 3. Werner J et al. Advanced-stage pancreatic cancer: therapy options. Nature Reviews Clinical Oncology, 2013 4. Hidalgo M et al. Translational therapeutic opportunities in ductal adenocarcinoma of the pancreas. Clin Cancer Res, 2012 5. Collisson EA et al. Subtypes of pancreatic ductal adenocarcinoma and their differing responses to therapy. Nat Med, 2011 6. Feig C et al. Targeting CXCL12 from FAP-expressing carcinoma-associated fibroblastssynergizes with anti-PD-L1 immunotherapy in pancreatic cancer. Proc Natl Acad Sci USA, 2013 7. Provenzano PP et al. Enzymatic targeting of the stroma ablates physical barriers to treatment of pancreatic ductal adenocarcinoma. Cancer Cell, 2012 8. Neesse A et al. Emerging concepts in pancreatic cancer medicine: targeting the tumor stroma. Onco Targets Ther, 2013 Citation Format: Elisa Espinet Espinet, Christian Eisen, Elisa M. Noll, Vanessa Vogel, Corinna Klein, Zuguang Gu, Matthias Schlesner, Tobias Bauer, Nathalia Giese, Roland Eils, Jens Werner, Wilko Weichert, Martin R. Sprick, Andreas Trumpp. Exploring the PDAC-subtype-associated microenvironment in PDX models and patients. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A61.

Abstract B59: Transnuclear mice as models of the immune response to pancreatic cancer

Abstract Somatic cell nuclear transfer (SCNT) allows the efficient generation of mice that will serve as a source of T cells of defined specificity. By harvesting primary T cells from animals that are at the peak of an anti-tumor response, and by transfer of the nucleus from such antigen specific T cells into an enucleated oocyte, embryonic stem cells that harbor the TCR rearrangements of that T cell may be obtained and used for the construction of cloned mice. These animals contain T cells of the appropriate tumor specificity, have no genetic alterations other than the physiological TCR rearrangements and are the closest approximation of the tumor-specific CTLs that contribute to tumor resolution in vivo. Importantly, the generation of transnuclear mice is rapid, requiring approximately 6 weeks from T cell harvest to obtaining chimeric animals. No good animal models are available for studying the immune response to pancreatic cancer, unlike in melanoma and prostate cancer for which we have excellent models. We are addressing this problem by generating a panel of transnuclear mice from pancreatic tumor infiltrating CD4 and CD8 T cells. These mice will 1) allow us to characterize the optimal CD8 and CD4 immune response necessary for rejection of pancreatic tumors; 2) define the spectrum of antigens seen by pancreatic tumor-infiltrating regulatory T cells; 3) address possible synergy of anti-tumor CD8 T cells with costimulatory blockade, silencing of anergy-related genes, or combination therapy with small molecule based chemotherapy; and 4) generate further interest in the community for studying immune-based therapies for pancreatic cancer. One of the best characterized pancreatic tumor antigens in both mice and humans is mesothelin. We have generated a TN mouse line from a mesothelin-specific CD8 T cell. However, the resulting mouse shows downregulation of CD8 in the thymus, and produces CD8 T cells that are functionally anergic. This anergy can be overcome by prolonged culture with anti-CD3/CD28 coated beads, and the resulting activated Mes358 TN cells accumulate in the pancreatic tumor environment and block the early stages of pancreatic tumor growth. As a second approach, we used CRISPR technology to generate mesothelin-/- mice. When immunized with mesothelin, these mice will provide a robust source of Mes358 tetramer+ CD8 T cells for use as a donor population for SCNT to generate a second line of mesothelin-specific TN mice with higher affinity for mesothelin. Immune-based therapies show a variable degree of success across different tumor types, with melanoma being highly amenable to anti-CTLA and other checkpoint blockade therapies while pancreatic cancer has been more refractory although combination of Ipilimumab with GVAX has shown modest efficacy. Many factors could contribute to this disparity, notably stroma and poor vascular density that limit drug delivery to pancreatic tumors. Understanding the requirements for CD8 T cell priming, trafficking and function in the context of stromal depletion is likely key to the success or failure of immunotherapies for pancreatic cancer. Citation Format: Stephanie K. Dougan, Angelina Bilate, Rudolf Jaenisch, Hidde L. Ploegh. Transnuclear mice as models of the immune response to pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B59.

Antitumor activity of mHSP65-TTL enhanced by administration of low dose cyclophosphamide in pancreatic cancer-bearing mice

Pancreatic cancer remains a lethal malignancy. Despite chemotherapy or/and radiotherapy after the surgery, the improvement on the overall survival of the patients has still been minimal. To develop novel therapeutic approaches, we tried to prepare mHSP65-TTL, a candidate vaccine prepared by mixing the recombinant mycobacterial heat shock protein 65 (mHSP65) with tumor tissue lysate (TTL) of Panc02 pancreatic cancer tissue. The mHSP65-TTL were used to immune the C57BL/6 mice implanted with the Panc02 cancer cells, in combination with or without low dose cyclophosphamide (CY). The results showed that mHSP65-TTL significantly prolonged the survival of the pancreatic cancer bearing mice and low dose CY enhanced the efficacy of the mHSP65-TTL. In addition, we detected mRNA expression of RORγt and IL-17A in spleen cells of mice received mHSP65-TTL or mHSP65-TTL plus CY, and found that mHSP65-TTL up-regulated mRNA expressions of RORγt and IL-17A, CY alone or mHSP65-TTL plus CY up-regulated mRNA expressions of RORγt. The work could provide an insight into a combinational approach for the immunotherapy of pancreatic cancer.

ITOC2 – 036. A method for developing predictive tests for immunotherapy benefit

Immunotherapies offer the promise of greatly improved outcomes, but often in only a minority of patients. It is of great importance to be able to identify which patients are likely to benefit from new immunotherapy advances. Developing tests to identify patients likely to benefit from immunotherapeutics presents special issues due to the lack of clear endpoints able to demonstrate which patients benefit from therapy. Traditional categorical endpoints, such as response, may not identify all patients who benefit and may be poor surrogates for long term progression-free survival and overall survival. Using methods adapted from the field of deep learning, we have developed an approach to multivariate test development for benefit from therapy able to work with the most clinically meaningful endpoints, by simultaneously refining the definition of relative benefit groups, the subset of most relevant genetic or proteomic information, and the diagnostic test itself. During the iterative process of test creation, the system learns what subset of the available molecular data is useful for the particular problem being addressed. We will demonstrate how this method has been used to develop a test predictive of benefit from an immunotherapy in adjuvant pancreatic cancer.

A multicenter randomized, open-label, proof-of-concept, phase II trial comparing gemcitabine with and without IMM-101 in advanced pancreatic cancer.

336 Background: Immunotherapy is becoming established as an effective way of treating cancer. Immodulon Therapeutics is developing IMM-101, a heat-killed whole cell preparation of Mycobacterium obuense (NCTC 13365), which modulates systemic immune responses, as an adjunctive immunotherapy for pancreatic cancer. Methods: Patients with advanced pancreatic cancer and a WHO score of 0-2 were assigned randomly to receive IMM-101 (0.1 mL intradermal injection of 10 mg/mL) plus Gemcitabine (Gem) (1000 mg/m2for 3 consecutive weeks out of 4) or Gem alone. Per protocol, this could be continued to a 12-cycle maximum. The primary efficacy endpoint was overall survival (OS). Safety, tolerability and progression free survival (PFS) were also assessed. Results: A total of 110 patients were randomized, 75 to receive IMM-101 plus Gem and 35 Gem alone (ITT population). The PP population consisted of 63 and 35 patients, respectively. Conclusions were similar; here we report results of the PP analysis. Median OS was increased significantly by 29% to 7.2 months in the IMM-101 plus Gem group compared to 5.6 months in the Gem group (p=0.022; HR 0.60, 95% CI 0.38-0.94). Median PFS was increased significantly by 83% to 4.4 months in the IMM-101 plus Gem group compared to 2.4 months in the Gem group (p=0.003; HR 0.51, 95% CI 0.32-0.81). In the pre-defined sub-group of patients with metastatic disease (n=82), median OS was increased significantly by 70% to 7.5 months in the IMM-101 plus Gem group (n=54) compared to 4.4 months in the Gem group (n=28) (p=0.002; HR 0.46, 95% CI 0.28-0.76). A highly significant 91% increase in median PFS from 2.3 months in the Gem group to 4.4 months in the IMM-101 plus Gem group was observed (p<0.001; HR 0.40, 95% CI 0.24-0.66). IMM-101 was well tolerated. The only adverse events of NCI CTC ≥Grade 3 occurring more frequently in the IMM-101 group (absolute difference in frequency between the groups >5%) were asthenia 10.8% and abdominal pain 8.1% (both 2.9% in the Gem group). Conclusion: Clinically meaningful increases in OS and PFS were demonstrated with IMM-101. No additional burden of adverse events above those relating to chemotherapy or the underlying disease was observed. Clinical trial information: NCT01303172.

Resistance of cyclooxygenase-2 expressing pancreatic ductal adenocarcinoma cells against γδ T cell cytotoxicity

The prostaglandin (PG) synthetase cyclooxygenase 2 (Cox-2) promotes tumorigenesis, tumor progression, and metastasis in a variety of human cancer entities including pancreatic ductal adenocarcinoma (PDAC). In this study, we demonstrate that in PDAC cells such as Colo357 cells, enhanced Cox-2 expression and increased release of the Cox-2 metabolite prostaglandin E2 (PGE2) promotes resistance against γδ T cell-mediated lysis. Co-culture with activated γδ T cells induced an upregulation of Cox-2 expression in Colo357 cells, and thereby an enhanced PGE2 release, in response to tumor necrosis factor α (TNFα) secretion from γδ T cells. The PGE2-mediated inhibition of γδ T cell cytotoxicity against Cox-2-expressing PDAC cells can be partially overcome by Cox-2 inhibitors. Our results show that differences between PDAC cells in regards to sensitivity to γδ T-cell cytotoxicity can be due to distinct levels of Cox-2 expression associated with varying amounts of PGE2 release. While γδ T cell cytotoxicity against PDAC cells expressing low levels of Cox-2 can be effectively enhanced by tribody [(Her2)2×Vγ9] with specificity for Vγ9 T cell receptor and HER-2/neu on PDAC cells, a combination of tribody [(Her2)2×Vγ9] and Cox-2 inhibitor is necessary to induce complete lysis of Cox-2 high expressing Colo357. In conclusion, our results suggest that the application of tribody [(Her2)2×Vγ9] that enhances γδ T-cell cytotoxicity and Cox-2 inhibitors that overcome PGE2-mediated resistance of PDAC cells to the cytotoxic activity of γδ T cells might offer a promising combined immunotherapy for pancreatic cancer.

Cancer immunotherapy for pancreatic cancer utilizing α-gal epitope/natural anti-Gal antibody reaction.

Pancreatic ductal adenocarcinoma (PDAC) has the poorest prognosis of all malignancies and is largely resistant to standard therapy. Novel treatments against PDAC are desperately needed. Anti-Gal is the most abundant natural antibody in humans, comprising about 1% of immunoglobulins and is also naturally produced in apes and Old World monkeys. The anti-Gal ligand is a carbohydrate antigen called "α-gal epitopes" with the structure Galα1-3Galβ1-4GlcNAc-R. These epitopes are expressed as major carbohydrate antigens in non-primate mammals, prosimians, and New World monkeys. Anti-Gal is exploited in cancer vaccines to increase the immunogenicity of antigen-presenting cells (APCs). Cancer cells or PDAC tumor lysates are processed to express α-gal epitopes. Vaccination with these components results in in vivo opsonization by anti-Gal IgG in PDAC patients. The Fc portion of the vaccine-bound anti-Gal interacts with Fcγ receptors of APCs, inducing uptake of the vaccine components, transport of the vaccine tumor membranes to draining lymph nodes, and processing and presentation of tumor-associated antigens (TAAs). Cancer vaccines expressing α-gal epitopes elicit strong antibody production against multiple TAAs contained in PDAC cells and induce activation of multiple tumor-specific T cells. Here, we review new areas of clinical importance related to the α-gal epitope/anti-Gal antibody reaction and the advantages in immunotherapy against PDAC.

A Novel Chimeric Antigen Receptor Against Prostate Stem Cell Antigen Mediates Tumor Destruction in a Humanized Mouse Model of Pancreatic Cancer

Despite advances in the understanding of its molecular pathophysiology, pancreatic cancer remains largely incurable, highlighting the need for novel therapies. We developed a chimeric antigen receptor (CAR) specific for prostate stem cell antigen (PSCA), a glycoprotein that is overexpressed in pancreatic cancer starting at early stages of malignant transformation. To optimize the CAR design, we used antigen-recognition domains derived from mouse or human antibodies, and intracellular signaling domains containing one or two T cell costimulatory elements, in addition to CD3zeta. Comparing multiple constructs established that the CAR based on human monoclonal antibody Ha1-4.117 had the greatest reactivity in vitro. To further analyze this CAR, we developed a human pancreatic cancer xenograft model and adoptively transferred CAR-engineered T cells into animals with established tumors. CAR-engineered human lymphocytes induced significant antitumor activity, and unlike what has been described for other CARs, a second-generation CAR (containing CD28 cosignaling domain) induced a more potent antitumor effect than a third-generation CAR (containing CD28 and 41BB cosignaling domains). While our results provide evidence to support PSCA as a target antigen for CAR-based immunotherapy of pancreatic cancer, the expression of PSCA on selected normal tissues could be a source of limiting toxicity.

Pancreatic cancer immunotherapy using a tumor lysate vaccine, engineered to express α-gal epitopes, targets pancreatic cancer stem cells.

Pancreatic cancer is a lethal disease that remains one of the most resistant to traditional therapies. Immunotherapy in pancreatic cancer induces the recruitment and activation of T cells that recognize tumor-associated antigens (TAAs); thus, the mechanism differs from that of chemotherapy and radiotherapy. The goal of cancer immunotherapy is to elicit immune responses against autologous tumors, and especially to induce multiple T cell clones against a variety of TAAs. In the present study, we prepared a polyvalent tumor lysate vaccine engineered to express the α-gal epitopes, Galα1-3Galβ1-4 GlcNAc-R (i.e., α-gal tumor lysate), from primary tumors. The vaccine elicited strong antibody production against multiple TAAs in pancreatic cancer cells and induced activation of multiple tumor-specific T cells in α1,3-galactosyltransferase (α1,3GT) knockout (KO) mice. The tumor lysate vaccine exhibited a similar effect on pancreatic cancer stem cells (CSCs) with the CD44+CD24+ phenotype. Furthermore, invivo experiments using NOD/SCID mice, inoculated with splenocytes from KO mice vaccinated with the α-gal tumor lysate and injected with pancreatic cancer cells, showed successful induction of a marked immune response that resulted in suppression of tumorigenesis and significant improvement in overall survival. In contrast, inoculation of lymphocytes from KO mice vaccinated with control tumor lysate vaccine had no effects on tumor growth and survival. The results of both invitro and invivo experiments emphasize the efficiency of tumor lysate vaccines expressing α-gal epitopes in targeting all pancreatic cancer cells, including differentiated cancer cells and pancreatic CSCs. The α-gal tumor lysate vaccine could be the basis for a novel therapeutic approach in human clinical trials.

Abstract SY26-01: Vaccine induced immunity against cancer genes to prevent and treat pancreatic cancer

Abstract Pancreatic cancer remains a “death sentence,” with the poorest survival rates reported for solid tumors. Escalating this problem is the fact that pancreatic cancer is among the most resistant cancers to traditional forms of therapy. But in this new era of targeted therapies, there is reason to hope that significant progress will be made in the development of new interventions for both treatment and prevention in the near future. In fact, we have entered into a new era of cancer therapies and are witnessing the rapid approval of antibodies and small molecule agents that inhibit cancer associated pathways. In the past few years we have also witnessed the approval of vaccines for cancer prevention (HPV vaccines, Gardasil, Cervarix) and cancer treatment (prostate cancer vaccine, Sipuleucel-T, Provenge); and the first immune modulating agent (Yervoy, Ipilimumab) that blocks the down regulatory signal, CTLA-4 on T cells, thereby enhancing immune activity against malignant melanoma. These groundbreaking examples have opened the floodgates for a whole new class of anticancer agents that harness the power of the immune system. Until recently, barriers to immunotherapy clinical successes for pancreatic cancer have been due to a lack of understanding of the immune pathways within the pancreatic tumor microenvironment that hinder successful immune responses even at the earliest stages of cancer development. Recent advances in our understanding of its immunobiology has identified new targets for developing cancer specific vaccines and new targets for modulating the tumor microenvironment to allow more potent activation and improved access of vaccine induced immune responses. We are just learning that non-neoplastic cells, including cancer-associated myofibroblasts, regulatory T cells, dendritic cells, myeloid-derived suppressor cells, and tumor-associated macrophages, are hijacked by pre-invasive and invasive cancer cells to create a tolerogenic tumor microenvironment. Current research is focused on elucidating the mechanisms of this cancer supporting polarization within the tumor microenvironment with the goal of tipping the balance in favor of an anticancer immunity. With the recent advances in molecular technologies and the development of relevant pancreatic cancer mouse models, it is now within our reach to dissect the inhibitory pathways within the pancreatic tumor microenvironment. This will in turn, lead to new therapeutic opportunities that will eliminate these barriers and convert this deadly cancer into a treatable and hopefully preventable disease. This presentation will provide new data supporting a role for targeting tumor initiating and metastasis supporting genes as immunotherapy for preventing tumor initiation and progression.

Abstract 2581: Pancreatic cancer immunotherapy with chitosan/IL-12

Abstract Pancreatic cancer is difficult to treat due to its late diagnoses and limited treatment options. Strategies capable of reducing tumor mass so that patients are eligible for tumor resection, i.e. the Whipple procedure, and strategies for reducing metastatic deposits with immunotherapy are needed. New immunotherapy techniques can provide an alternative to modern chemotherapy, radiation, or surgical options which have not been able to reduce pancreatic cancer mortality. IL-12 has been discovered to be a potent agent in the development of anti-tumor immunity through its ability to stimulate the activities of cytotoxic T lymphocytes, NK cells, and macrophages while promoting Th1 differentiation. We have previously discovered that a bioadhesive chitosan/IL-12 combination can aggressively eliminate implanted tumors in murine models. Chitosan is a non-toxic and degradable polysaccharide derived from crustacean shells that shows great potential for use in immunotherapy models due to its unique binding properties. Here, ability of chitosan/IL-12 vaccinations to slow the growth of a pancreatic adenocarcinoma cells in vivo was investigated. C57BL/6 mice received subcutaneous inoculations with Panc02 cells. After Panc02 tumors became visible, the mice received 4 weekly intratumoral immunotherapy treatments with either saline or chitosan/IL-12. There was a prominent delay in the tumor growth of the chitosan/IL-12 group at all stages of the treatment. Seven days after the second treatment, control mice had an average tumor volume of 180mm3 and treatment mice tumors became undetectable. Seven days after the fourth treatment, the control mice had an average tumor volume of 743mm3 and treatment mice had an average tumor volume of 221mm3. Cell proliferation assays showed that IL-12 had no direct effect on the Panc02 cell line. However, interferon-γ, a cytokine whose production is induced by IL-12, reduced the growth of Panc02 cells in vitro. These data shows that chitosan/IL-12 may be an effective immunotherapy for pancreatic cancer and also that IL-12 is not directly cytotoxic, but rather, it initiates an effector response capable of inhibiting pancreatic tumors. Citation Format: Haley K. Perlow, Lirong Yang, David Zaharoff. Pancreatic cancer immunotherapy with chitosan/IL-12. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2581. doi:10.1158/1538-7445.AM2014-2581