Gene Therapy (2016) 23,38–49 © 2016 Macmillan Publishers Limited All rights reserved 0969-7128/16 www.nature.com/gt ORIGINAL ARTICLE Mesothelin-specific cell-based vaccine generates antigen- specific immunity and potent antitumor effects by combining with IL-12 immunomodulator M-C Chang1,2, Y-L Chen3, Y-C Chiang1, T-C Chen1, Y-C Tang1, C-A Chen1, W-Z Sun2 and W-F Cheng1,4,5 Ovarian cancer is a gynecologic malignancy with a high mortality rate. In the present study, we developed a novel cell-based vaccine, Meso-VAX, to generate mesothelin antigen-specific immune responses and immunotherapy against ovarian cancer. Mesothelin, a secreted protein anchored at the cell membrane, has recently been identified as a potential new tumor antigen for ovarian cancer. In this study, mice vaccinated with Meso-VAX and adeno-associated virus (AAV)-IL-12 exhibited dramatic increases in the number of mesothelin-specific CD4+ helper and CD8+ cytotoxic T-cell precursors, higher titers of anti-mesothelin Abs and in vitro tumor killing activity, and all of these mice were tumor-free after 60 days of tumor challenge. In addition, a significant reduction in peritoneal tumors and longer survival were noted in the mice vaccinated with Meso-VAX combined with AAV-IL-12. CD4+ helper and CD8+ cytotoxic T lymphocytes were essential for the antitumor effect generated by Meso-VAX combined with AAV-IL-12. The post-vaccination sera of the mice vaccinated with Meso-VAX and AAV-IL-12 also showed mesothelin-specific complement-dependent cell-mediated cytotoxicity. Our results suggest that a Meso-VAX cell-based vaccine combined with AAV- IL-12 can generate antigen-specific immunological responses and antitumor effects on ovarian cancer. Gene Therapy (2016) 23, 38–49; doi:10.1038/gt.2015.85 INTRODUCTION histocompatibility complex) class I and class II-restricted 14 Ovarian cancer has one of the highest mortality rates among epitopes. The main disadvantage of autologous and allogeneic gynecologic malignancies.1 Around 75% of patients were diag- whole-cell tumor vaccines is their limited ability to stimulate an nosed at an advanced stage in the 1980s,2,3 and the overall immune response. However, the best method to enhance host survival (OS) rate was only 19–30% from 1955 to 1993.2,3 The immunity to generate effective antitumor responses with whole- standard treatment is surgical tumor debulking, followed by cell tumor vaccines remains unresolved. platinum-based chemotherapy.4,5 Although platinum-based Mesothelin is a secreted protein anchored on the cell 15 chemotherapy results in a response rate of around 80% in all membrane by a glycosylphosphatidylinositol linkage. Mesothe- stages of ovarian cancer,6 50–70% of patients experience lin is absent or present at low levels in normal ovarian tissues, but 16,17 recurrence and ultimately succumb to the malignancy.7 The highly-expressed in ovarian cancer cells, and Hassan et al. 15 development of novel drugs and new therapeutic strategies for suggested that it can be highly immunogenic. In a previous ovarian cancer are therefore urgently needed. study, mesothelin was found to inhibit paclitaxel-induced When developing an effective therapy for ovarian cancer, a apoptosis through a PI3K (phosphoinositide 3-kinase)/Akt-depen- strategy is needed to attack cancer cells only without destroying dent pathway, suggesting that mesothelin can reduce the normal cells and to avoid the development of drug resistance. chemotherapeutic sensitivity of ovarian cancer.17 Mesothelin has Immunotherapy meets these criteria, and it could be used as an also been reported to be a potential tumor marker for the adjuvant to surgery or in combination with chemotherapy or progression of gastric15,16,18,19 and ovarian cancers,17 and it has other biologic therapy such as chemo-immunotherapy or also been identified in triple-negative breast cancer patients.20 bio-chemo-immunotherapy. Thus, mesothelin represents a potential target antigen for the The identification of tumor-associated antigens (TAAs) and host development of immunotherapy against different types of cancer immunity stimulation are essential steps in the development of a such as malignant mesothelioma, gastric, ovarian and breast cancer vaccine.8,9 Various formulations have been investigated as cancers. sources of TAAs for vaccinations, including defined peptides or The tumor cell-based vaccine WF-0 was originally derived from TAAs derived from the entire tumor proteome,10 and whole-cell C57BL/6 mouse peritoneal cells transduced with human papilloma tumor vaccines have been investigated for decades.11–13 virus type 16 E6 and E7 genes to generate immortalized clones. Allogeneic or autologous tumor cells are processed (lysates or The immortalized peritoneal cells were further transfected with irradiated cells) to optimize the release of their antigens by the activated c-Ha-ras gene and then further injected into athymic providing a full complement of TAAs, including MHC (major nude mice, which subsequently developed peritoneal-based 1Department of Obstetrics and Gynecology, Medicine College of Medicine, National Taiwan University, Taipei, Taiwan; 2Department of Anesthesiology, Medicine College of Medicine, National Taiwan University, Taipei, Taiwan; 3Department of Obstetrics and Gynecology, Cathay General Hospital, Taipei, Taiwan; 4Graduate Institute of Clinical Medicine, Medicine College of Medicine, National Taiwan University, Taipei, Taiwan and 5Graduate Institute of Oncology, Medicine College of Medicine, National Taiwan University, Taipei, Taiwan. Correspondence: Dr W-F Cheng, Department of Obstetrics and Gynecology, National Taiwan University Hospital, 7, Chung-Shan South Road, Taipei 100, Taiwan. E-mail: [email protected] Received 15 November 2014; revised 12 June 2015; accepted 23 June 2015; accepted article preview online 11 August 2015; advance online publication, 17 September 2015 Antigen-specific cell-based immunotherapy M-C Chang et al 39 tumors and ascites. The isolated tumor cells from athymic mice bearing mice. The post-vaccination sera of the mice vaccinated were called WF-0, and a previous study confirmed that WF-0 cells with Meso-VAX and AAV-IL-12 also showed enhanced mesothelin- could not generate tumors in immunocompetent mice.21 specific complement-dependent cytotoxicity. Furthermore, there Moreover, due to a lack of endogenous mesothelin expression, were significant preventive and therapeutic effects to inhibit other genes can be introduced into WF-0 cells to generate tumor growth and prolong survival in the mice vaccinated with possible ovarian cancer antigen-specific immune responses and Meso-VAX and AAV-IL-12. These results indicate that the antigen- possible antitumor effects. Thus, WF-0 cells could be an ideal specific cell-based vaccine, Meso-VAX, can enhance the TAA, tumor cell-based vaccine to investigate potential ovarian cancer mesothelin, and specific T cell-mediated immunity and antitumor immunotherapy strategies. effects when combined with the IL-12 cytokine. Tumor-associated A mesothelin-expressing ascitogenic malignant tumor model cell-based vaccines combined with AAV-IL-12 may be able to (WF-3) demonstrating morphological features of intra-peritoneal break through immune tolerance, and represent an innovative tumorigenesis has been developed.21 In this model, the WF-3 approach in the development of cancer immunotherapy for tumor cells express high levels of mesothelin, commonly observed ovarian cancer. in intra-peritoneal tumors such as ovarian cancer and malignant mesothelioma. Thus, the WF-3 tumor model would serve as an excellent pre-clinical tumor model to study clinically relevant RESULTS issues such as the screening and evaluation of the effectiveness of Ectopic-expressing human mesothelin in murine ovarian cancer therapeutic interventions by monitoring the level of mesothelin. cells To date, whole-cell (including autogenic or allogenic) vaccines RT-PCR and flow-cytometry analysis showed that the Meso-VAX have been designed to induce specific antitumor immune tumor cells expressed higher levels of human mesothelin RNA responses.22,23 However, the application of cell-based vaccines is (Figure 1a) and protein (Figure 1b) compared with the paternal limited because only a small number of TAAs has been identified. WF-0 and WF-0/mock cells. This indicated that WF-0 cells Another problem is that tumor cells may escape from the immune transfected with human mesothelin gene could express human response due to immunological editing. Therefore, mesothelin as mesothelin. a potential TAA for ovarian cancer can be expected to generate immune tolerance in the host. Vaccination with Meso-VAX and AAV-IL-12 enhanced the In this study, we introduced the human mesothelin gene into mesothelin-specific cell-mediated and humoral immunity WF-3 tumor cells to generate the xenogenic antigen-specific, The representative figures of flow-cytometry analysis of cell-based vaccine Meso-VAX. The combination of Meso-VAX mesothelin-specific interferon (IFN)-γ-secreting CD4+ helper and adeno-associated virus (AAV)-IL-12 was further tested to T-cell precursors in the various groups (Figure 2a) showed investigate whether this strategy could break immune tolerance that the mice vaccinated with Meso-VAX and AAV-IL-12 to generate antitumor effects and enhance both mesothelin- (0.051 ± 0.0014%) had a significantly higher percentage of specific cellular and humoral immunity. Vaccination with mesothelin-specific IFN-γ-secreting CD4+ T-cell precursors Meso-VAX and AAV-IL-12 enhanced the number of mesothelin- compared with the other groups (PBS: 0.003 ± 0.0007%; specific CD4+ helper and CD8+ T cytotoxic cell precursors and WF-0: 0.003 ± 0.0008%; WF-0/mock: 0.005 ± 0.0012%; Meso-VAX: generated higher titers of anti-mesothelin Abs in WF-3 tumor 0.003 ± 0.0005%; AAV-IL-12: 0.009 ± 0.0007%; WF-0 with AAV-IL-12: Figure 1. Characterization of human mesothelin expression in Meso-VAX cells. (a) RNA expression levels of mesothelin in various WF-0 transfectants by RT-PCR. Note: The mesothelin expression was significantly increased in the Meso-VAX cells compared with the parental WF-0 and WF-0/mock cells.