Immunotherapy Of Ovarian Cancer Research Articles

Abstract A37: Epigenetic modification of ovarian cancer immunogenicity

Abstract Background: Ovarian high-grade serous carcinoma (HGSC) remains a poor prognosis disease due to its late presentation and lack of “actionable” mutations to target pharmacologically. The host immune system, however, plays a pivotal role with the presence of cytotoxic T lymphocytes and chemokines that regulate T-cell trafficking both having positive effect on survival in preclinical and clinical studies. HGSC cells can achieve immune escape via epigenetic silencing of key immune-related genes, including via DNA methylation; when DNA methylation is pharmacologically reversed, genes involved in antigen presentation, receptor-dependent immune cell stimulation, and chemokine release are reactivated. One of these genes encodes the angiostatic chemokine Cxcl10, which is a T-lymphocyte attractant. In order to elucidate other potential epigenetic mechanisms directly or indirectly involved in Cxcl10 gene silencing, we will screen a library of novel epigenetic probes that target bromodomains, methyltransferases, demethylases, and others epigenetic pathways (Structural Genomic Consortium library). Aims: We will use Trp53-/- ID8 ovarian cancer cells to perform a medium-throughput screening of 40 novel epigenetic probes, measuring cxcl10 production, with the demethylating drug decitabine as positive control. Methods: Trp53-/- ID8 cells were treated with decitabine for up to 72 hours. Cell-cycle analysis was assessed via BrdU incorporation assay. Cytokine/chemokine transcription was assessed using RT2 profiler 84 gene chemo/cytokine PCR array (Qiagen), and verified using qRT-PCR. Acid-hydrolysed DNA was subjected to mass spectrometry/liquid chromatography to quantify the mean ratio of methyl-cytosine/cytosine. Western immunoblotting was performed to test DNMT1 protein levels in protein lysates after decitabine treatment. Cxcl10 levels in supernatant were quantified by ELISA. Results: Treatment of Trp53-/- ID8 cells with decitabine (DAC), at non-cytostatic/cytotoxic doses causes upregulation of multiple chemokines, specifically Cxcl10. 200nM DAC for 48 hours increased Cxcl10 transcription 22-fold (95% CI 20.6-21.1-fold, p=0.0001, values normalized to Gapdh control), and protein levels in supernatant three-fold (t test p<0.0001). At this dose, DNTM1, the target enzyme of DAC, is downregulated on Western-blot analysis, and the methyl-cytosine/cytosine ratio decreases significantly (means compared with t test, p =0.002). Other statistically significant upregulated chemokine genes are Cxcl1 (p=0.002), Cxcl3 (p<0.001), Cxcl4 (p<0.001), Cxcl5 (p=0.03), and Ccl4 (p=0.01). Conclusions: Abrogation of DNA methylation with DAC in Trp53-/- ID8 cells induces an increase in the chemokine cxcl10, at both transcriptional and protein level. This provides a robust control for a medium-throughput screening of other epigenetic modifiers that are still at an early drug development stage, such as probes against bromodomains, methyltransferases, and demethylases. Such screening can unmask novel epigenetic mechanisms involved in immune-related gene transcription and offer new ways to potentiate immunotherapy in ovarian cancer. Citation Format: Pavlina Spiliopoulou, Josephine Walton, Suzanne Dowson, Alex Binks, Oliver Maddocks, Peter Adams, Iain McNeish. Epigenetic modification of ovarian cancer immunogenicity. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A37.

Abstract IA22: Reprogramming the tumor microenvironment and T cells for ovarian cancer immunotherapy

Abstract Immunologic destruction of tumors is a multistep, coordinated process that can be modulated or targeted at several critical points to elicit tumor rejection. These steps in the cancer immunity cycle include: (i) generation of sufficient numbers of effector T cells with high avidity recognition of tumor antigens in vivo; (ii) trafficking and infiltration into the tumor; (iii) overcoming inhibitory networks in the tumor microenvironment; (iv) direct recognition of tumor antigens and generation of an effector antitumor response; and (v) persistence of the antitumor T cells. Although several immune-based interventions have gained regulatory approval in many solid tumors and hematologic malignancies, there are currently no approved immune therapies for ovarian cancer. A major barrier to successful deployment of cancer immunotherapy for ovarian cancer patients is the immunosuppressive tumor microenvironment. Even if large numbers of tumor-specific T cells are generated in patients by active immunization or adoptive transfer, these T cells may not persist to readily destroy tumor targets in vivo. Previous studies by our group have defined some of the dominant immune resistance mechanisms in ovarian cancer to include extrinsic suppression of CD8+ effector cells by regulatory T cells (Tregs) (1, 2); metabolic deregulation via tryptophan catabolism by the immunoregulatory enzyme indoleamine-2,3-dioxygenase (IDO) (3, 4); engagement of inhibitory receptors such as PD-1 by the ligand PD-L1/B7-H1 (5-7); development of antigen loss variants (8); myeloid-derived suppressor cells (9); and inhibitory cytokines such as TGF-β. Collectively, this redundant immunosuppressive network facilitates tumor progression by actively restraining antitumor immunity and must be overcome in order to implement efficacious immunotherapeutic strategies. This presentation will be focused on our efforts at: (i) overcoming IDO-mediated innate and adaptive immune resistance as a strategy to reprogram the ovarian tumor microenvironment and (ii) novel “next-generation” strategies for reprograming T cells for adoptive immunotherapy of ovarian cancer.

Understanding of targeting MyD88, IDO1 and AHR at the heart of immunosuppressive signaling pathway for immunotherapy of epithelial ovarian cancer

Understanding of targeting MyD88, IDO1 and AHR at the heart of immunosuppressive signaling pathway for immunotherapy of epithelial ovarian cancer

Abstract 4567: Immunomodulatory action of SGI-110 and combination with γδ T cell immunotherapy in ovarian cancer

Abstract Ovarian cancer (OC) is regarded as being an immunogenic cancer and strategies to enhance immune function in patients are thought to confer benefit and improve prognosis. Immunotherapy has been shown to be most promising when combined with other therapeutic approaches. Recent evidence in the field and preliminary data from our group suggest that treatment with epigenetic drugs, such as DNA methyltransferase inhibitors (DNMTi), can lead to improved immune activation in several cancers including ovarian. The purpose of this study is to test the hypothesis that treatment with next generation hypomethylating agent SGI-110 can induce the re-expression of immunomodulatory genes and endogenous retroviruses (ERVs), which shifts OC cells towards a more immunogenic expression profile. OC cell lines SKOV3, Kuramochi and OVSAHO were treated with low dose SGI-110 and assessed for increased immunogenicity. Expression of immune biomarkers and ERVs was assessed at different time points at mRNA levels by qPCR and protein levels by Flow Cytometry. Treated cell lines were functionally tested for increased immunogenicity by co-culture with γδ T cells, which were isolated from healthy donors and expanded by zoledronic acid selection. MTT assays were performed to assess cell viability and T cell killing following co-culture. IFNγ and Granzyme B release from T cells and chemokine release from tumor cells were assessed by ELISA. Our results show up-regulation of key immunogenicity biomarkers and ERVs in our panel of OC cell lines, following treatment with SGI-110. Co-culturing SGI-110-treated cell lines with γδ T cells resulted in increased tumor cell killing and T cell activity. We are now analysing RNA-sequencing data of SGI-110-treated OC cell lines in an effort to dissect the immunomodulatory mechanism of action of this novel hypomethylating agent. Moreover, we plan to test the in vivo feasibility of a SGI-110/γδ T cell combination therapy, which if successful might be further optimised for a clinical trial. Citation Format: Marina Natoli, Anastasios Karadimitris, Sadaf Ghaem-Maghami. Immunomodulatory action of SGI-110 and combination with γδ T cell immunotherapy in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4567.

Oncolytic virus immunotherapies in ovarian cancer: moving beyond adenoviruses.

Ovarian cancer is the 5th most common cancer in UK women with a high relapse rate. The overall survival for ovarian cancer has remained low for decades prompting a real need for new therapies. Recurrent ovarian cancer remains confined in the peritoneal cavity in >80% of the patients, providing an opportunity for locoregional administration of novel therapeutics, including gene and viral therapy approaches. Immunotherapy is an expanding field, and includes oncolytic viruses as well as monoclonal antibodies, immune checkpoint inhibitors, and therapeutic vaccines.Oncolytic viruses cause direct cancer cell cytolysis and immunogenic cell death and subsequent release of tumor antigens that will prime for a potent tumor-specific immunity. This effect may be further enhanced when the viruses are engineered to express, or coadministered with, immunostimulatory molecules. Currently, the most commonly used and well-characterized vectors utilized for virotherapy purposes are adenoviruses. They have been shown to work synergistically with traditional chemotherapy and radiotherapy and have met with success in clinical trials. However, pre-existing immunity and poor in vivo models limit our ability to fully investigate the potential of oncolytic adenovirus as effective immunotherapies which in turn fosters the need to develop alternative viral vectors. In this review we cover recent advances in adenovirus-based oncolytic therapies targeting ovarian cancer and recent advances in mapping immune responses to oncolytic virus therapies in ovarian cancer.

Targeting vitamin D receptor (VDR)/immune checkpoint inhibitor receptor ligand PD-L1 axis for immunotherapy of ovarian cancer

Objective: VDR is highly over-expressed in epithelial ovarian cancer (EOC) and correlates with worse survival rate of patients. Cause and pathologic impact of VDR over-expression in EOC are not known. Our ongoing studies indicate that VDR expression activates PD-L1, which promotes immune evasion in tumors via deactivation of cytotoxic T-cells. We propose to study outcome of VDR/PD-L1 interactions and to determine whether targeting VDR/PD-L1 axis via a novel VDR antagonist MeTC7 can restore antitumor immunity and reduce tumor burden.

Complement system's role in cancer and its therapeutic potential in ovarian cancer.

Cancer immunotherapy is a strong candidate for the long-awaited new edition to standard cancer therapies. For an effective immunotherapy, it is imperative to delineate the players of antitumour immune response. As an important innate immune system effector mechanism, complement is highly likely to play a substantial role in cancer immunity. Studies suggest that there may be two different "states of complement" that show opposing effects on cancer cells; a complement profile that has antitumour effects with low expression of membrane-bound complement regulator proteins (mCRPs), lytic membrane attack complex (MAC) concentration and moderate C5a concentration, and a complement profile that has protumour effects with high expression of mCRPs, sublytic MAC and high concentrations of C5a. One of the cancers that urgently require innovative therapeutic approaches is ovarian cancer, and complement has a potential to be a good target for this purpose. A combinatorial approach where the complement cascade is fine-tuned by inhibiting some of its activities while promoting the others can prove to be a fruitful approach. Herein, we will briefly discuss the cancer-immune system interaction and then present a discussion of complement system's role in tumour immunity and its therapeutic potential for ovarian cancer immunotherapy.

Preclinical development of a therapeutic antibody for the treatment of epithelial ovarian carcinoma

Background: Despite active research in ovarian cancer immunotherapy, standard of care for ovarian cancer remains surgery and (neo)adjuvant chemotherapy. Patients often develop secondary resistance to chemotherapy, leading to recurrence. Since most mouse models have had a poor translational efficiency so far, there is a need for new therapy development strategies. We here present a mostly human-based pipeline that we have used for target identification and drug development and testing.

Slow-Release Formulation of Cowpea Mosaic Virus for In Situ Vaccine Delivery to Treat Ovarian Cancer.

The plant viral nanoparticle cowpea mosaic virus (CPMV) is shown to be an effective immunotherapy for ovarian cancer when administered as in situ vaccine weekly, directly into the intraperitoneal (IP) space in mice with disseminated tumors. While the antitumor efficacy is promising, the required frequency of administration may pose challenges for clinical implementation. To overcome this, a slow release formulation is developed. CPMV and polyamidoamine generation 4 dendrimer form aggregates (CPMV‐G4) based on electrostatic interactions and as a function of salt concentration, allowing for tailoring of aggregate size and release of CPMV. The antitumor efficacy of a single administration of CPMV‐G4 is compared to weekly administration of soluble CPMV in a mouse model of peritoneal ovarian cancer and found to be as effective at reducing disease burden as more frequent administrations of soluble CPMV; a single injection of soluble CPMV, does not significantly slow cancer development. The ability of CPMV‐G4 to control tumor growth following a single injection is likely due to the continued presence of CPMV in the IP space leading to prolonged immune stimulation. This enhanced retention of CPMV and its antitumor efficacy demonstrates the potential for viral–dendrimer hybrids to be used for delayed release applications.

Immunotherapy in Ovarian Cancer

Immunotherapy in Ovarian Cancer

Natural Killer Cell-Based Immunotherapy in Gynecologic Malignancy: A Review.

Harnessing the immune system has proven an effective therapy in treating malignancies. Since the discovery of natural killer (NK) cells, strategies aimed to manipulate and augment their effector function against cancer have been the subject of intense research. Recent progress in the immunobiology of NK cells has led to the development of promising therapeutic approaches. In this review, we will focus on the recent advances in NK cell immunobiology and the clinical application of NK cell immunotherapy in ovarian, cervical, and uterine cancer.

High Immune Expression of Progesterone-Induced Blocking Factor in Epithelial Ovarian Cancer.

Background:Progesterone-induced blocking factor, which is released from maternal lymphocytes during pregnancy mediates the immune effect of progesterone. According to new reports, it is suggested that proliferating cells, such as human trophoblasts, mesenchymal stem cells, and malignant tumors, can excrete progesterone-induced blocking factor at high ratio to escape from maternal immunity. It is shown in recent studies that progesterone-induced blocking factor is overexpressed in many malignant tumors such as breast, cervical, lymphoma, and leukemia. There are no data about progesterone-induced blocking factor expression in ovarian cancer cells. Hence, it is aimed to determine the progesterone-induced blocking factor expression levels in epithelial ovarian cancer.Methods:The study which was a retrospective cross-sectional study was conducted in a University Hospital. Twenty tissue specimens of patients with epithelial ovarian cancer and 20 tissue specimens of patients with healthy ovary were included in the study. Primary rabbit polyclonal anti- progesterone-induced blocking factor antibody was used to incubate the sections at a ratio of 1:300.Results:When the tissue sections were compared based on immunostaining with progesterone-induced blocking factor, we detected high stromal progesterone-induced blocking factor expression in the epithelial ovarian cancer group as check against to the normal ovarian group (P = .007). Similarly, we found high glandular progesterone-induced blocking factor expression in the epithelial ovarian cancer group as check against to the normal ovarian group (P < .001).Conclusion:Proving the existence of progesterone-induced blocking factor expression in epithelial ovarian cancer cells may lead new visions or new studies for epithelial ovarian cancer immunotherapy. As a result, epithelial ovarian cancer cells have greater levels of expression of progesterone-induced blocking factor protein than normal ovarian tissue according to immunohistochemistry. Further research is needed to understand the clinical importance of this finding, to learn outcomes of high levels of progesterone-induced blocking factor, and to investigate its underlying mechanisms.

Clinical significance of promoter hypermethylation of genes in ovarian cancer

// Xinxin Zhu 1, 2 and Jinghe Lang 1 1 Department of Obstetrics and Gynecology, Peking Union Medical College Hospital, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, China 2 Department of Obstetrics and Gynecology, Institute for Wound Research, University of Florida, Gainesville, Florida, USA Correspondence to: Jinghe Lang, email: langjh@hotmail.com , doctorlangjh@163.com Keywords: epigenetic; DNA methylation; gene silencing; immunotherapy; ovarian cancer Received: August 02, 2017&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Accepted: December 18, 2017&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Published: December 29, 2017 ABSTRACT Ovarian cancer is the most lethal condition in gynecologic oncology. The known prognostic factors for ovarian cancer include tumor stage, histological grade, lymph node, and distant metastasis; however, handy and reliable molecular biomarkers for prevention, diagnosis, personalized treatment, and prognosis are scarce. Despite histological differences, the clinical treatment strategy is similar for ovarian cancers. The survival rate for ovarian cancer, however, remains relatively low. Accumulating evidence suggests that hypermethylated promoters of genes can be promising candidates as molecular biomarkers in ovarian cancer risk evaluation, early detection, personalized treatment, and prognosis. With advancements in immunology, hypermethylation of gene promoters was found to alter the tumor immune microenvironment, and gene methyltransferase inhibitors can contribute to ovarian cancer immunotherapy by boosting tumor immunogenicity and immune response and decreasing immunosuppression. Although DNMTis demonstrate high efficacy in hematologic malignancies, the application of DNMTis in solid tumors is just in its beginning. This article, drawing on both preclinical and clinical data, systematically reviews the common hypermethylated genes in ovarian cancer and their clinical applications, evaluating their usefulness in early diagnosis, personalized treatment, prognosis prediction, and the establishment of combined therapy of methyltransferase inhibitors and immunotherapy.

Immunotherapy in ovarian cancer

Immunological destruction of tumors is a multistep, coordinated process that can be modulated or targeted at several critical points to elicit tumor rejection. These steps in the cancer immunity cycle include: (i) generation of sufficient numbers of effector T cells with high avidity recognition of tumor antigens in vivo; (ii) trafficking and infiltration into the tumor; (iii) overcoming inhibitory networks in the tumor microenvironment; (iv) direct recognition of tumor antigens and generation of an effector anti-tumor response; and (v) persistence of the anti-tumor T cells. In an effort to understand whether the immune system plays a role in controlling ovarian cancer, our group and others demonstrated that the presence of tumor infiltrating lymphocytes (TILs) is associated with improved clinical outcome in ovarian cancer patients. Recently, we hypothesized that the quality of infiltrating T cells could also be a critical determinant of outcome in ovarian cancer patients. In the past decade, several immune-based interventions have gained regulatory approval in many solid tumors and hematologic malignancies. These interventions include immune checkpoint blockade, cancer vaccines, and adoptive cell therapy. There are currently no approved immune therapies for ovarian cancer. Immunotherapy in ovarian cancer will have to consider the immune suppressive networks within the ovarian tumor microenvironment; therefore, a major direction is to develop biomarkers that would predict responsiveness to different types of immunotherapies, and allow for treatment selection based on the results. Moreover, such biomarkers would allow rational combination of immunotherapies, while minimizing toxicities. In this review, the current understanding of the host immune response in ovarian cancer patients will be briefly reviewed, progress in immune therapies, and future directions for exploiting immune based strategies for long lasting durable cure.

2014

OBJECTIVES/SPECIFIC AIMS: The specific objective of this proposal is to identify and validate targetable tumor-associated antigens (TAAs) in ovarian and pancreatic cancer. It is our central hypothesis that the accurate identification and selection of appropriate TAAs will provide a foundation on which to develop of novel and effective cancer immunotherapies. We have formulated this hypothesis on the basis of preliminary results in which we have used high-throughput tandem mass spectrometry (MS) to successfully identify TAAs from melanoma patient tumors. We have subsequently generated TAA-specific T-cells that showed specific recognition and killing of tumor cells, and will form the basis of an upcoming clinical trial for our melanoma patients. We now have extended this antigen identification pipeline into ovarian cancer to accomplish our objective of developing effective T-cell-based immunotherapies for ovarian cancer and pancreatic patients. METHODS/STUDY POPULATION: We have collected patient tumor specimens, and we performed HLA immunoprecipitation, peptide elution, and completed high-throughput tandem MS on these eluted samples to identify TAAs. In addition, we have validated the safety of potential targets through the use of the publicly available RNA sequence data sources GTEX and TCGA. RESULTS/ANTICIPATED RESULTS: To date, we have successfully completed over 60 peptide elutions from ovarian and pancreatic patients samples. In total, we have found several potential novel tumor-associated targets. VGLL1, is one of these identified antigens, and in conjunction with our collaborators, we have successfully generated T-cells against it. Additionally, we have found that VGLL1 is a potential novel TAA for 3 other cancer types, including bladder, gastric, and triple negative breast cancers. We are now focusing our efforts on testing these T-cells against additional ovarian cancer cell lines and these cancer types to determine their specificity. We plan to continue the generation and testing other identified potential TAAs as well. We plan to use these T-cells directly in clinical trials in the future. DISCUSSION/SIGNIFICANCE OF IMPACT: The rationale for this proposal is that through the identification and validation of TAAs, we can open the door to a new world of therapies that can potentially increase the survival rate in a disease with a historically grim prognosis.

Abstract 2793: Evidence for WT1 as a potential target for immunotherapy of lethal ovarian cancer

Abstract Epithelial ovarian cancer is the leading cause of death from gynecologic cancers with 22,280 new cases anticipated in 2016 and 14,240 deaths. Most patients are diagnosed at late stages and have a poor prognosis. Radical surgery and chemotherapy are the primary treatment. When relapse occurs there are few treatment options. The overall survival rate of women with ovarian cancer is unchanged in 50 years. Approximately 70% of epithelial ovarian cancers are serous including low grade serous tumors, some of which may evolve from serous borderline tumors, and high grade serous carcinomas. The latter are thought to originate in the fallopian tube. There are eight other subtypes of invasive ovarian cancer. All invasive ovarian cancers have recently been classified into two subtypes, Type 1 (indolent) and Type 2 (aggressive). The Wilms’ Tumor 1 (WT1) protein is expressed in some normal tissues including the fallopian tube and ovary as well as in tumors such as ovarian and breast cancers and in leukemia. WT1 is a highly immunogenic tumor associated antigen. Mutated p53 is found in many high grade ovarian cancers. Recent reports indicate that WT1 and p53 may interact physically and functionally. Both WT1 and mutated p53 have been suggested as prognostic markers in ovarian cancer. We sought to determine the role of co-expression of mutated p53 and WT1 in overall survival of patients with ovarian cancer. Histologic sections were prepared from archived FFPE surgical specimens donated by St. Elizabeth Healthcare (Northern KY) and fully annotated by hospital records. H&amp;E stained sections were diagnosed and graded by a board certified pathologist (LED) and included histologic sections from 41 borderline tumors and 98 Type 1 and Type 2 cancers. Adjacent sections were stained immunohistochemically using monoclonal antibodies (DAKO) to WT1 and p53. For survival analysis p53 was considered mutated if 75-100% of the tumor nuclei were stained (missense mutations) or if all nuclei were negative (truncated protein) and WT1 was considered either non-expressed (no stain) or expressed (some or all nuclei stained). While either mutated p53 or WT1 were prognostic alone of patient survival with Type 1 and Type 2 tumors, WT1 was a better predictor as seen by log-rank Chi-square. The 20 year survival probability of patients with tumors negative for both mutated p53 and WT1 was 70% and was significantly better than other patients (p &amp;lt; 0.0001). The 20 year survival probability for tumors negative for WT1 but expressing mutated p53 was 41%, and was 9% for tumors expressing WT1 and non-mutated p53. Patients with tumors expressing both WT1 and mutated p53 had a 20 year survival probability of 6%. Given these data and the immunogenicity of the WT1 tumor associated antigen we conclude that IHC detection of mutated p53 and WT1 in invasive ovarian cancers would be useful in stratifying patients eligible for immunologic approaches targeting WT1 for therapy of lethal ovarian cancers. Citation Format: Julia H. Carter, James A. Deddens, Gretchen Mueller, Thomas G. Lewis, Mariah K. Dooley, Jackson O. Pemberton, Larry E. Douglass. Evidence for WT1 as a potential target for immunotherapy of lethal ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2793. doi:10.1158/1538-7445.AM2017-2793

Abstract 3649: Combination of mesothelin-targeted immune-activating fusion protein and anti-PD-L1 augments antitumor immunity and prolongs survival in murine model of ovarian cancer

Abstract Background and Purpose: Although immunotherapy as an adjuvant to surgery and chemotherapy has been investigated in ovarian cancer (OC) as a means of reducing tumor recurrence and improving survival, there remains a significant unmet need for combinatorial strategies to enhance the antitumor immune response. The purpose of this study was to develop a novel combination immunotherapy for OC, utilizing our novel fusion protein to target and generate a cellular immune response to mesothelin (MSLN) in conjunction with blockade of the PD-1/PD-L1 checkpoint to restore the function of cytotoxic T cells in order to enhance cancer control and prolong survival. Experimental Procedures: Luciferase-expressing ID8 cells were employed to establish an intraperitoneal ovarian tumor model in immunocompetent C57BL/6 mice. The efficacies of the MSLN-targeted immune-activating fusion protein (VIC-008), αPD-L1, and the combination were evaluated. Mice received 4 intraperitoneal (i.p.) treatments of VIC-008 from day 7 post tumor inoculation weekly, and 6 treatments of αPD-L1 i.p. every other day from 4 weeks post inoculation. Tumor growth was monitored by in vivo imaging of luciferase activity. Survival time was calculated as life span from the day of tumor inoculation. In immunological studies, mice were sacrificed 7 weeks after tumor cell inoculation. Immune cells from lymph nodes, ascites and tumors were stained with antibodies against multiple immune cell markers and profiled by flow cytometry. Results: VIC-008, αPD-L1 or combination treatment delayed tumor growth. The combination treatment resulted in the greatest prolongation in survival, followed by αPD-L1 treatment and then VIC-008 treatment. Improved survival was associated with increased levels of intratumoral CD3+CD8+ T cells (P&amp;lt;0.0001). The combination treatment also reduced the proportion of CD4+CD25+Foxp3+ Treg cells (P&amp;lt;0.0001) in the lymph nodes. An increased number of CD8+CD27+CD44+ memory T cells (P=0.0134) were observed in ascites in the combination treatment group. CD11b+CD11c+ dendritic cells were enriched in ascites in VIC-008 treatment (P=0.0019) and combination treatment groups (P=0.0010). More CD11c+CD38+ (M1) (P=0.0361) and fewer CD206+CD106+ (M2) (P=0.0285) macrophages were found in the tumors of the combination treatment group. Conclusion: Our results suggest that, through activating dendritic cells and enhancing antigen presentation and cross-presentation, VIC-008 augments antitumor CD8+ T cell responses and facilitates generation of memory T cells when combined with PD-1/PD-L1 blockade, providing long-term antitumor effects. Our findings demonstrate for the first time a mechanistic rationale for combining VIC-008 and αPD-L1 in treatment of OC in mice, positioning this combination therapy as a potential promising new immunotherapeutic approach for OC. Citation Format: Yang Zeng, Binghao Li, Qiuyan Liu, Patrick Reeves, Ann Sluder, Jeffrey Gelfand, Timothy Brauns, Mark Poznansky, Huabiao Chen. Combination of mesothelin-targeted immune-activating fusion protein and anti-PD-L1 augments antitumor immunity and prolongs survival in murine model of ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3649. doi:10.1158/1538-7445.AM2017-3649

Abstract 1608: CD122-selective IL-2/anti-IL-2 complexes reduce regulatory T cell function and promote CD8+ T cell polyfunctionality for durable ovarian cancer immunotherapy

Abstract The IL-2 receptor (IL-2R) is an attractive target for cancer immunotherapy as it controls both immune-suppressive regulatory T cells (Tregs) and anti-tumor T cells. We tested depleting Tregs as immunotherapy using anti-CD25 (high-affinity IL-2R subunit) antibodies (αCD25) in ID8agg mouse ovarian cancer (OC). αCD25 reduced ascites and Treg numbers but failed to reduce tumor burden, possibly because it depleted newly activated anti-tumor T cells in tumor-draining lymph nodes. Thus, αCD25 could be novel malignant ascites palliation, but has limited stand-alone efficacy. We then tested IL-2/anti-IL-2 complexes (IL-2c) that selectively stimulate medium-affinity (CD122/CD132) IL-2R thought to expand anti-tumor T cells preferentially, but with little Treg effects. In contrast to several single agents we tested that failed to treat ID8agg (e.g., αCD25, αPD-L1, IL-2 fusion toxin denileukin diftitox), IL-2c alone durably reduced ID8agg tumor burden despite lowering the tumor microenvironmental CD8+/Treg ratio. Thus, we hypothesized that IL-2c improved CD8+ function, reduced Treg function, or both. IL-2c increased polyfunctional IFN-γ+TNF-α+ anti-tumor T cells as expected, an effect that persists weeks after drug clearance. IL-2c also increased anti-tumor T cell CD25 expression that increased IL-2 sensitivity and STAT5 phosphorylation, a likely mechanism for increased polyfunctionality. Unexpectedly, IL-2c reduced the Treg functional mediators CD25, TIGIT and granzyme B, and reduced Treg suppressive function. Thus, favorable Treg modifications are a novel IL-2c mechanism of action. Adding αCD25 to IL-2c to deplete Tregs further unexpectedly worsened IL-2c efficacy in ID8agg and reduced effector memory T cells and polyfunctional T cells in the tumor microenvironment, suggesting a previously unappreciated role for CD25 in IL-2c therapy. Similar data were seen in B16 melanoma, suggesting αCD25 reduction of IL-2c efficacy is not tumor or compartment-specific (ID8agg is peritoneal and B16 is subcutaneous). αPD-L1, an ineffective monotherapy in ID8agg, combined with IL-2c to promote complete responses, suggesting potential for potent, novel combinatorial approaches. Our data suggest that antagonizing high affinity IL-2R (such as to deplete Tregs with αCD25) has limited cancer immunotherapy utility without more specific Treg targeting. In contrast, stimulating medium-affinity IL-2R with CD122-selective IL-2c has great translational promise by simultaneously improving beneficial anti-tumor T cells and reducing detrimental Treg function. Citation Format: Justin M. Drerup, Sri Lakshmi Pandeswara, Aijie Liu, Curtis A. Clark, Alvaro S. Padron, Wanjiao Chen, Vincent Hurez, Tyler J. Curiel. CD122-selective IL-2/anti-IL-2 complexes reduce regulatory T cell function and promote CD8+ T cell polyfunctionality for durable ovarian cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1608. doi:10.1158/1538-7445.AM2017-1608

Novel target for ovarian cancer immunotherapy

Novel target for ovarian cancer immunotherapy

The safety, efficacy, and treatment outcomes of a combination of low-dose decitabine treatment in patients with recurrent ovarian cancer

ABSTRACTPurpose: DNA demethylating agents have shown clinical effectiveness in hematological and solid tumors. This trial tested the safety, efficacy, and treatment outcomes of decitabine-based chemotherapy or combined with immunotherapy in recurrent ovarian cancer patients.Patients and methods: Fifty-five patients with recurrent ovarian cancer were enrolled and 52 were assessable for clinical response and survival. Patients either received 5-d decitabine treatment, followed by reduced-dose of paclitaxel/carboplatin administration (DTC cohort), or the aforementioned regimen combined with cytokine-induced killer cells therapy (DTC+CIK cohort). The primary end point was clinical response rate and progression-free survival (PFS). Secondary evaluation included safety assessment and overall survival (OS).Results: Disease control rate (DCR) and objective response rate (ORR) were 73.91% and 23.91% in disease measurable patients by RECIST criteria, totally 76.92% and 30.77%, including disease non-measurable patients, which were higher in platinum-resistant/refractory patients. Clinical benefits could be associated with the number of DAC treatment cycles and the inclusion of CIK immunotherapy. In DTC+CIK cohort, DCR and ORR reached 100% and 58.30%, respectively. Notably, DTC+CIK treatment in platinum-resistant/refractory patients had an ORR of 87.50%. Consistently, PFS was longer in platinum-resistant/refractory patients comparing with that of platinum-sensitive patients. PFS and OS were 8 and 19 mo in platinum-resistant/refractory patients with DTC+CIK therapy. The most common toxicities were nausea, anorexia, fatigue, neutropenia, and anemia; many of which were grade 1–2.Conclusion: Low-dose DAC/paclitaxel/carboplatin regimen demonstrates disease benefit, especially in patients with platinum-resistant/refractory ovarian cancer, and might show remarkable clinical response when combined with adoptive immunotherapy in platinum-resistant/refractory ovarian cancer patients.