Th17-inducing dendritic cell vaccines stimulate effective CD4 T cell-dependent antitumor immunity in ovarian cancer that overcomes resistance to immune checkpoint blockade.

BACKGROUND: Ovarian cancer (OC), a highly lethal cancer in women, has a 48% 5-year overall survival rate. Prior studies link the presence of IL-17 and Th17 T cells in the tumor microenvironment to improved survival in OC patients. To determine if Th17-inducing vaccines are therapeutically effective in OC, we created a murine model of Th17-inducing dendritic cell (DC) (Th17-DC) vaccination generated by stimulating IL-15 while blocking p38 MAPK in bone marrow-derived DCs, followed by antigen pulsing. METHODS: ID8 tumor cells were injected intraperitoneally into mice. Mice were treated with Th17-DC or conventional DC (cDC) vaccine alone or with immune checkpoint blockade (ICB). Systemic immunity, tumor associated immunity, tumor size and survival were examined using a variety of experimental strategies. RESULTS: Th17-DC vaccines increased Th17 T cells in the tumor microenvironment, reshaped the myeloid microenvironment, and improved mouse survival compared with cDC vaccines. ICB had limited efficacy in OC, but Th17-inducing DC vaccination sensitized it to anti-PD-1 ICB, resulting in durable progression-free survival by overcoming IL-10-mediated resistance. Th17-DC vaccine efficacy, alone or with ICB, was mediated by CD4 T cells, but not CD8 T cells. CONCLUSIONS: These findings emphasize using biologically relevant immune modifiers, like Th17-DC vaccines, in OC treatment to reshape the tumor microenvironment and enhance clinical responses to ICB therapy.

Characterization of Morreton virus as an oncolytic virotherapy platform for liver cancers.

BACKGROUND: Morreton virus (MORV) is an oncolytic Vesiculovirus , genetically distinct from vesicular stomatitis virus (VSV). AIM: To report that MORV induced potent cytopathic effects (CPEs) in cholangiocarcinoma (CCA) and hepatocellular carcinoma (HCC) in vitro models. APPROACH AND RESULTS: In preliminary safety analyses, high intranasal doses (up to 10 10 50% tissue culture infectious dose [TCID 50 ]) of MORV were not associated with significant adverse effects in immune competent, non-tumor-bearing mice. MORV was shown to be efficacious in a Hep3B hepatocellular cancer xenograft model but not in a CCA xenograft HuCCT1 model. In an immune competent, syngeneic murine CCA model, single intratumoral treatments with MORV (1 × 10 7 TCID 50 ) triggered a robust antitumor immune response leading to substantial tumor regression and disease control at a dose 10-fold lower than VSV (1 × 10 8 TCID 50 ). MORV led to increased CD8 + cytotoxic T cells without compensatory increases in tumor-associated macrophages and granulocytic or monocytic myeloid-derived suppressor cells. CONCLUSIONS: Our findings indicate that wild-type MORV is safe and can induce potent tumor regression via immune-mediated and immune-independent mechanisms in HCC and CCA animal models without dose limiting adverse events. These data warrant further development and clinical translation of MORV as an oncolytic virotherapy platform.

Repurposing live attenuated trivalent MMR vaccine as cost-effective cancer immunotherapy.

It has long been known that oncolytic viruses wield their therapeutic capability by priming an inflammatory state within the tumor and activating the tumor immune microenvironment, resulting in a multifaceted antitumor immune response. Vaccine-derived viruses, such as measles and mumps, have demonstrated promising potential for treating human cancer in animal models and clinical trials. However, the extensive cost of manufacturing current oncolytic viral products makes them far out of reach for most patients. Here by analyzing the impact of intratumoral (IT) administrations of the trivalent live attenuated measles, mumps, and rubella viruses (MMR) vaccine, we unveil the cellular and molecular basis of MMR-induced anti-cancer activity. Strikingly, we found that IT delivery of low doses of MMR correlates with tumor control and improved survival in murine hepatocellular cancer and colorectal cancer models via increased tumor infiltration of CD8+ granzyme B+ T-cells and decreased macrophages. Moreover, our data indicate that MMR activates key cellular effectors of the host's innate and adaptive antitumor immunity, culminating in an immunologically coordinated cancer cell death. These findings warrant further work on the potential for MMR to be repurposed as safe and cost-effective cancer immunotherapy to impact cancer patients globally.

Myxoma virus lacking the host range determinant M062 stimulates cGAS-dependent type 1 interferon response and unique transcriptomic changes in human monocytes/macrophages.

The evolutionarily successful poxviruses possess effective and diverse strategies to circumvent or overcome host defense mechanisms. Poxviruses encode many immunoregulatory proteins to evade host immunity to establish a productive infection and have unique means of inhibiting DNA sensing-dependent type 1 interferon (IFN-I) responses, a necessity given their dsDNA genome and exclusively cytoplasmic life cycle. We found that the key DNA sensing inhibition by poxvirus infection was dominant during the early stage of poxvirus infection before DNA replication. In an effort to identify the poxvirus gene products which subdue the antiviral proinflammatory responses (e.g., IFN-I response), we investigated the function of one early gene that is the known host range determinant from the highly conserved poxvirus host range C7L superfamily, myxoma virus (MYXV) M062. Host range factors are unique features of poxviruses that determine the species and cell type tropism. Almost all sequenced mammalian poxviruses retain at least one homologue of the poxvirus host range C7L superfamily. In MYXV, a rabbit-specific poxvirus, the dominant and broad-spectrum host range determinant of the C7L superfamily is the M062R gene. The M062R gene product is essential for MYXV infection in almost all cells tested from different mammalian species and specifically inhibits the function of host Sterile α Motif Domain-containing 9 (SAMD9), as M062R-null (ΔM062R) MYXV causes abortive infection in a SAMD9-dependent manner. In this study we investigated the immunostimulatory property of the ΔM062R. We found that the replication-defective ΔM062R activated host DNA sensing pathway during infection in a cGAS-dependent fashion and that knocking down SAMD9 expression attenuated proinflammatory responses. Moreover, transcriptomic analyses showed a unique feature of the host gene expression landscape that is different from the dsDNA alone-stimulated inflammatory state. This study establishes a link between the anti-neoplastic function of SAMD9 and the regulation of innate immune responses.

Th17-inducing autologous dendritic cell vaccination promotes antigen-specific cellular and humoral immunity in ovarian cancer patients.

In ovarian cancer (OC), IL-17-producing T cells (Th17s) predict improved survival, whereas regulatory T cells predict poorer survival. We previously developed a vaccine whereby patient-derived dendritic cells (DCs) are programmed to induce Th17 responses to the OC antigen folate receptor alpha (FRα). Here we report the results of a single-arm open-label phase I clinical trial designed to determine vaccine safety and tolerability (primary outcomes) and recurrence-free survival (secondary outcome). Immunogenicity is also evaluated. Recruitment is complete with a total of 19 Stage IIIC-IV OC patients in first remission after conventional therapy. DCs are generated using our Th17-inducing protocol and are pulsed with HLA class II epitopes from FRα. Mature antigen-loaded DCs are injected intradermally. All patients have completed study-related interventions. No grade 3 or higher adverse events are seen. Vaccination results in the development of Th1, Th17, and antibody responses to FRα in the majority of patients. Th1 and antibody responses are associated with prolonged recurrence-free survival. Antibody-dependent cell-mediated cytotoxic activity against FRα is also associated with prolonged RFS. Of 18 patients evaluable for efficacy, 39% (7/18) remain recurrence-free at the time of data censoring, with a median follow-up of 49.2 months. Thus, vaccination with Th17-inducing FRα-loaded DCs is safe, induces antigen-specific immunity, and is associated with prolonged remission.

Gastrointestinal Tract Dysbiosis Enhances Distal Tumor Progression through Suppression of Leukocyte Trafficking.

The overall use of antibiotics has increased significantly in recent years. Besides fighting infections, antibiotics also alter the gut microbiota. Commensal bacteria in the gastrointestinal tract are crucial to maintain immune homeostasis, and microbial imbalance or dysbiosis affects disease susceptibility and progression. We hypothesized that antibiotic-induced dysbiosis of the gut microbiota would suppress cytokine profiles in the host, thereby leading to changes in the tumor microenvironment. The induced dysbiosis was characterized by alterations in bacterial abundance, composition, and diversity in our animal models. On the host side, antibiotic-induced dysbiosis caused elongated small intestines and ceca, and B16-F10 melanoma and Lewis lung carcinoma progressed more quickly than in control mice. Mechanistic studies revealed that this progression was mediated by suppressed TNFα levels, both locally and systemically, resulting in reduced expression of tumor endothelial adhesion molecules, particularly intercellular adhesion molecule-1 (ICAM-1) and a subsequent decrease in the number of activated and effector CD8+ T cells in the tumor. However, suppression of ICAM-1 or its binding site, the alpha subunit of lymphocyte function-associated antigen-1, was not seen in the spleen or thymus during dysbiosis. TNFα supplementation in dysbiotic mice was able to increase ICAM-1 expression and leukocyte trafficking into the tumor. Overall, these results demonstrate the importance of commensal bacteria in supporting anticancer immune surveillance, define an important role of tumor endothelial cells within this process, and suggest adverse consequences of antibiotics on cancer control. SIGNIFICANCE: Antibiotic-induced dysbiosis enhances distal tumor progression by altering host cytokine levels, resulting in suppression of tumor endothelial adhesion molecules and activated and effector CD8+ T cells in the tumor.

A comparison of alum sludge with peat for aqueous glyphosate removal for maximizing their value for practical use.

This study compares and contrasts the glyphosate removal efficiency of alum sludge (waterworks residue) and Irish peat in aqueous solution. Organic phosphonate of glyphosate aqueous solution was removed in pot tests separately filled with peat and alum sludge, while effluent samples were taken from each pot to analyse the concentration of phosphorus (P) and COD (chemical oxygen demand); physical and chemical analysis for both media before and after use was carried out subsequently. The results show that the P removal capacity of alum sludge was significant (>99%), while the removal capacity of peat was considerably less than 10% after 10 weeks. Both materials significantly reduced the levels of COD, but it was noted that peat had a marginally greater initial P removal capacity (68 ± 22%) and did perform better than alum sludge (57 ± 12%). Moreover, pre-treatment is a crucial step to harness the full potential of peat. Overall, this study provides a scientific clue for sorbents selection when considering alum sludge and peat to maximize their value in practice.

Regulatory T Cells in Gynecologic Cancer.

Increasing evidence supports that regulatory T cells (Tregs) within the tumor, tumor draining lymph nodes, ascites and peripheral blood of patients with cancer are associated with poor prognosis. Tregs are important mediators of active immune evasion in cancer. In this review, the potential mechanisms of Treg actions and the roles of Tregs specifically in the tumor microenvironment derived from three types of gynecological cancers, cervical, vulvar and ovarian, are described. The correlations between Tregs and clinical immunotherapeutic study outcomes are discussed. Successful modulation of Tregs would likely have significant impact on the effectiveness of immunotherapeutic treatments in cancer patients.

IL10 Release upon PD-1 Blockade Sustains Immunosuppression in Ovarian Cancer.

Ligation of programmed cell death-1 (PD-1) in the tumor microenvironment is known to inhibit effective adaptive antitumor immunity. Blockade of PD-1 in humans has resulted in impressive, durable regression responses in select tumor types. However, durable responses have been elusive in ovarian cancer patients. PD-1 was recently shown to be expressed on and thereby impair the functions of tumor-infiltrating murine and human myeloid dendritic cells (TIDC) in ovarian cancer. In the present work, we characterize the regulation of PD-1 expression and the effects of PD-1 blockade on TIDC. Treatment of TIDC and bone marrow-derived dendritic cells (DC) with IL10 led to increased PD-1 expression. Both groups of DCs also responded to PD-1 blockade by increasing production of IL10. Similarly, treatment of ovarian tumor-bearing mice with PD-1 blocking antibody resulted in an increase in IL10 levels in both serum and ascites. While PD-1 blockade or IL10 neutralization as monotherapies were inefficient, combination of these two led to improved survival and delayed tumor growth; this was accompanied by augmented antitumor T- and B-cell responses and decreased infiltration of immunosuppressive MDSC. Taken together, our findings implicate compensatory release of IL10 as one of the adaptive resistance mechanisms that undermine the efficacy of anti-PD-1 (or anti-PD-L1) monotherapies and prompt further studies aimed at identifying such resistance mechanisms. Cancer Res; 77(23); 6667-78. ©2017 AACR.

Myxoma Virus Optimizes Cisplatin for the Treatment of Ovarian Cancer In Vitro and in a Syngeneic Murine Dissemination Model.

A therapeutic approach to improve treatment outcome of ovarian cancer (OC) in patients is urgently needed. Myxoma virus (MYXV) is a candidate oncolytic virus that infects to eliminate OC cells. We found that in vitro MYXV treatment enhances cisplatin or gemcitabine treatment by allowing lower doses than the corresponding IC50 calculated for primary OC cells. MYXV also affected OC patient ascites-associated CD14+ myeloid cells, one of the most abundant immunological components of the OC tumor environment; without causing cell death, MYXV infection reduces the ability of these cells to secrete cytokines such as IL-10 that are signatures of the immunosuppressive tumor environment. We found that pretreatment with replication-competent but not replication-defective MYXV-sensitized tumor cells to later cisplatin treatments to drastically improve survival in a murine syngeneic OC dissemination model. We thus conclude that infection with replication-competent MYXV before cisplatin treatment markedly enhances the therapeutic benefit of chemotherapy. Treatment with replication-competent MYXV followed by cisplatin potentiated splenocyte activation and IFNγ expression, possibly by T cells, when splenocytes from treated mice were stimulated with tumor cell antigen ex vivo. The impact on immune responses in the tumor environment may thus contribute to the enhanced antitumor activity of combinatorial MYXV-cisplatin treatment.

The glycosyltransferase GnT-III activates Notch signaling and drives stem cell expansion to promote the growth and invasion of ovarian cancer.

Glycosylation changes associated with cellular transformation can facilitate the growth and progression of tumors. Previously we discovered that the gene Mgat3 encoding the glycosyltransferase GnT-III is elevated in epithelial ovarian carcinomas (EOCs) and leads to the production of abnormal truncated N-linked glycan structures instead of the typical bisected forms. In this study, we are interested in discovering how these abnormal glycans impact the growth and progression of ovarian cancer. We have discovered using stable shRNA gene suppression that GnT-III expression controls the expansion of side-population cells, also known as cancer stem cells. More specifically, we found that GnT-III expression regulates the levels and activation of the heavily glycosylated Notch receptor involved in normal and malignant development. Suppression of GnT-III in EOC cell lines and primary tumor-derived cells resulted in an inhibition of Notch signaling that was more potent than pharmacologic blockage of Notch activation via γ-secretase inhibition. The inhibition resulted from the redirection of the Notch receptor to the lysosome, a novel mechanism. These findings demonstrate a new role for bisecting glycosylation in the control of Notch transport and demonstrate the therapeutic potential of inhibiting GnT-III as a treatment for controlling EOC growth and recurrence.

Signaling Circuits and Regulation of Immune Suppression by Ovarian Tumor-Associated Macrophages.

The barriers presented by immune suppression in the ovarian tumor microenvironment present one of the biggest challenges to development of successful tumor vaccine strategies for prevention of disease recurrence and progression following primary surgery and chemotherapy. New insights gained over the last decade have revealed multiple mechanisms of immune regulation, with ovarian tumor-associated macrophages/DC likely to fulfill a central role in creating a highly immunosuppressive milieu that supports disease progression and blocks anti-tumor immunity. This review provides an appraisal of some of the key signaling pathways that may contribute to immune suppression in ovarian cancer, with a particular focus on the potential involvement of the c-KIT/PI3K/AKT, wnt/ß-catenin, IL-6/STAT3 and AhR signaling pathways in regulation of indoleamine 2,3-dioxygenase expression in tumor-associated macrophages. Knowledge of intercellular and intracellular circuits that shape immune suppression may afford insights for development of adjuvant treatments that alleviate immunosuppression in the tumor microenvironment and enhance the clinical efficacy of ovarian tumor vaccines.

Defining and managing expectations for early immunotherapy cancer trials.

This review discusses the concept of expectations in assessing direct benefit to participants in phase I immunotherapy studies. With the push toward a faster assessment of clinical benefit or efficacy, limiting phase I studies to safety determination only is now viewed as obsolete and has been replaced by designs that draw attention to therapeutic benefit or efficacy. While this approach is touted as being more flexible in trial conduct, these designs are particularly problematic for immunotherapy studies. Defining and managing expectations is paramount on understanding the key axioms that emerge that include i) understanding bias in models and mechanistic results, ii) that no test is perfect, iii) it is difficult to select a good predictive biomarker in the absence of clinical data, even for targeted therapies, iv) markers predictive for monotherapy may not be predictive for combination therapy, and v) all about improved patient selection. Considering the heterogeneity of cancers and the immune response of the host, we think that immunotherapy should be developed in parallel with the identification of different clinico-pathological models of immune response to cancer. This approach would accomplish two important goals: 1) provide a biological understanding of the complete in vivo environment, thereby giving investigators the opportunity to optimize and maximize the effect of a specific immunotherapy agent and 2) addressing host environment issues simultaneously so that safety data and perceived benefit can be achieved more quickly.

Galectin-3 inhibition suppresses drug resistance, motility, invasion and angiogenic potential in ovarian cancer.

OBJECTIVE: Ovarian cancer is the most deadly gynecologic malignancy worldwide. Since the pathogenesis of ovarian cancer is incompletely understood, and there are no available screening techniques for early detection, most patients are diagnosed with advanced, incurable disease. In an effort to develop innovative and effective therapies for ovarian cancer, we tested the effectiveness of Galecti-3C in vitro. This is a truncated, dominant negative form of Galectin-3, which is thought to act by blocking endogenous Galectin-3. METHODS: We produced a truncated, dominant-negative form of Galectin-3, namely Galetic-3C. Ovarian cancer cell lines and primary cells from ovarian cancer patients were treated with Galectin-3C, and growth, drug sensitivity, and angiogenesis were tested. RESULT: We show, for the first time, that Galectin-3C significantly reduces the growth, motility, invasion, and angiogenic potential of cultured OC cell lines and primary cells established from OC patients. CONCLUSIONS: Our findings indicate that Galectin-3C is a promising new compound for the treatment of ovarian cancer.

Ovarian tumor ascites CD14+ cells suppress dendritic cell-activated CD4+ T-cell responses through IL-10 secretion and indoleamine 2,3-dioxygenase.

The observation that Th17 infiltration in ovarian cancer correlates with markedly improved survival has prompted the question of whether ovarian tumor antigen-specific Th17 responses could be stimulated by tumor vaccination. Dendritic cells (DCs) treated with IL-15 and an inhibitor of p38 MAPK signaling (DC(IL-15/p38inhib)) bias T-cell responses toward a Th1/Th17 phenotype, raising the prospect of therapeutic vaccination; however, significant barriers remain. Tumor vaccines, including DC vaccination, usually stimulate immune responses, but the lack of clinical responses in cancer patients has been disappointing. Possible reasons may include an inability of antitumor T cells to migrate into the tumor microenvironment, and an inability of T cells to retain effector function in the face of tumor-associated immune suppression. We found that ovarian tumor antigen-specific CD4(+) T cells induced by DC(IL-15/p38inhib) migrated in response to CXCL12 and CCL22 (both highly expressed in ovarian cancer) and to ascites CD14(+) myeloid cells. Cocultures showed that ascites CD14(+) cells markedly suppressed antigen-specific CD4(+) T responses, but suppression could be alleviated by treatment with anti-IL-10 or inhibition of indoleamine 2,3-dioxygenase. These results suggest that the efficacy of DC vaccination against ovarian cancer may be boosted by agents that inhibit tumor-associated CD14(+) myeloid cell suppression or indoleamine 2,3-dioxygenase activity.

Dendritic cell vaccination, immune regulation, and clinical outcomes in ovarian cancer.

Clinical optimism for dendritic cell vaccination against ovarian cancer has been tempered by the knowledge that tumors avail themselves of multiple mechanisms of immune evasion, thus blunting the efficacy of therapeutic vaccination. Mechanisms of immune suppression include infiltration by regulatory T cells (Treg) and myeloid suppressor cell populations, expression of co-inhibitory receptors, and expression of indoleamine 2,3-dioxygenase (IDO). Expression of both B7-H1 and IDO are associated with differentiation and recruitment of Treg, and clinical studies have shown that each of these mechanisms correlates independently with increased morbidity and mortality in ovarian cancer patients. In sharp contrast, recent studies have indicated that Th17 cell infiltration in ovarian cancer correlates with improved patient outcomes and prolonged overall survival. Given that IDO plays a pivotal role in the balance between Treg and Th17 immunity, elucidation of the mechanisms that regulate IDO activity and immune suppression may lead to novel adjuvants to boost the clinical efficacy of dendritic cell vaccination against ovarian cancer and other malignancies.

Modulation of p38 MAPK signaling enhances dendritic cell activation of human CD4+ Th17 responses to ovarian tumor antigen.

The recent finding that Th17 infiltration of ovarian tumors positively predicts patient outcomes suggests that Th17 responses play a protective role in ovarian tumor immunity. This observation has led to the question of whether Th17 cells could be induced or expanded to therapeutic advantage by tumor vaccination. In this study, we show that treatment of ovarian tumor antigen-loaded, cytokine-matured human dendritic cells (DC) with a combination of IL-15 and a p38 MAP kinase inhibitor offers potent synergy in antagonism of CD4(+) Treg induction and redirection toward CD4(+) Th17 responses that correlate with strong CD8(+) cytotoxic T lymphocyte (CTL) activation. Ovarian tumor antigen-specific CD4(+) T cells secrete high levels of IL-17 and show reduced expression of CTLA-4, PD-1, and Foxp3 following activation with IL-15/p38 inhibitor-treated DC. We further show that modulation of p38 MAPK signaling in DC is associated with reduced expression of B7-H1 (PD-L1), loss of indoleamine 2,3-dioxygenase activity, and increased phosphorylation of ERK 1/2 MAPK. These observations may allow the development of innovative DC vaccination strategies to boost Th17 immunity in ovarian cancer patients.

The case for HER2/neu as a therapeutic target for gynecologic malignancies.

Evaluation of: Guzzo F, Bellone S, Buza N et al. HER2/neu as a potential target for immunotherapy in gynecological carcinosarcomas. Int. J. Gynecol. Pathol. 31, 211-221 (2012). Trastuzumab (Herceptin(®)) is a human monoclonal antibody that is US FDA-approved for the treatment of HER2/neu-overexpressing breast cancer and metastatic gastric cancer. HER2/neu expression has also been observed in other malignancies, notably in subsets of endometrial and ovarian cancers, raising the possibility that trastuzumab may be a viable treatment option in these settings. The current paper reveals that HER2/neu may also be expressed in gynecologic carcinosarcomas, which are rare but aggressive tumors with a high rate of resistance to chemotherapy. Cell lines derived from ovarian carcinosarcomas expressed HER2/neu and were sensitive to trastuzumab ADCC. By contrast, cell lines derived from uterine carcinosarcomas were HER2/neu-negative and insensitive to trastuzumab ADCC. These observations indicate that at least a subset of gynecologic carcinosarcoma patients may benefit from trastuzumab treatment. However, trastuzumab monotherapy typically has relatively low response rates, which in part may be related to impaired NK cell function in patients with advanced disease. Accordingly, treatments that boost NK cell activity may improve response rates to trastuzumab treatment, not only for gynecologic carcinosarcomas, but also for other HER2/neu-positive malignancies.