Multiple Mechanisms Of Immune Suppression Research Articles

Neratinib Alone or in Combination with Immune Checkpoint Inhibitors with or without Mammalian Target of Rapamycin Inhibitors in Patients with Fibrolamellar Carcinoma

<b><i>Introduction:</i></b> Fibrolamellar carcinoma (FLC) displays upregulation of several oncogenes, including <i>HER2</i>, and multiple immune-suppressive mechanisms. We investigated the efficacy and safety of the pan-HER tyrosine kinase inhibitor neratinib as monotherapy (SUMMIT phase 2 basket study) or with immune checkpoint and/or mammalian target of rapamycin (mTOR) inhibitors (compassionate-use program) in patients with FLC. <b><i>Methods:</i></b> Patients received neratinib 240 mg/day orally in SUMMIT, or as doublet or triplet combinations with pembrolizumab 2 mg/kg intravenously every 3 weeks, nivolumab 240 mg intravenously every 2 weeks, everolimus 7.5 mg/day orally, or sunitinib 37.5 mg/day orally under compassionate use. The primary endpoint in SUMMIT was objective response rate; safety was a secondary endpoint. <b><i>Results:</i></b> Fifteen patients with FLC received neratinib monotherapy in SUMMIT. The objective response rate was 5% (95% confidence interval [CI]: 0–21.8) and the disease control rate was 13.3% (95% CI: 1.7–40.5). Upon progression, five had added immune checkpoint inhibitors with or without everolimus or sunitinib. Two additional patients received neratinib-based combinations outside of SUMMIT, for a total of 17 neratinib-treated patients. One patient who received neratinib plus pembrolizumab had a confirmed partial response, one treated with neratinib plus everolimus had stable disease lasting 6 months, and one who received neratinib plus pembrolizumab plus sunitinib had stable disease lasting 16 months. Grade 3/4 adverse events with neratinib monotherapy occurred in 10 (66.7%)/2 (13.3%) patients, respectively. Grade 3 adverse events with neratinib-based combinations were hyperglycemia (<i>n</i> = 1; neratinib plus pembrolizumab), hepatic failure, and anaphylaxis (<i>n</i> = 1 each, neratinib plus pembrolizumab plus everolimus). There were no grade 4 adverse events with combination therapy. <b><i>Conclusion:</i></b> In patients with FLC, single-agent neratinib had limited efficacy, but clinical benefit was observed with neratinib in combination with immunotherapy and/or mTOR-targeted agents.

Abstract 4523: Inhibition of arginase in combination with anti-PDL1 leads to increased infiltration and activation of CD8+ T cells, NK cells, and CD103+ dendritic cells in mouse syngeneic tumor models

Abstract The tumor microenvironment (TME) has multiple mechanisms of immune-suppression, one being recruitment of arginase (ARG) expressing myeloid cells. ARG is an enzyme that catalyzes hydrolysis of L-arginine into urea and L-ornithine. L-arginine is a semi-essential amino acid required for optimal function of T cells and natural killer (NK) cells. Arginine depletion in the TME inhibits T cell and NK cell function. Therefore, inhibition of ARG can reverse immune-suppression and optimize anti-tumor immunity. To determine the dependence to arginine, we cultured human T- and NK cells in arginine concentrations ranging from 0 to 150 µM. Both human CD8+ T cells and NK cells showed decreased proliferation with decreased L-arginine concentration, and a complete proliferation arrest in the absence of L-arginine. L-arginine threshold for optimal proliferation was determined to be ~30 µM for CD8+ T cells and ~ 9 µM for NK cells. Furthermore, both CD8+ T cells and NK cells showed decreases in cytotoxic molecules (e.g. granzyme A, granzyme B, perforin) when cultured under reduced arginine conditions. In addition, NK cells showed a decreased ability to kill target cells in low arginine conditions. We next explored whether arginase inhibition could reverse the immune-suppressive environment in vivo. Administration of arginase inhibitor to tumor bearing mice resulted in a dose dependent increase in both plasma (up to 4 fold) and tumor arginine (up to 12 fold) in all models tested including MC38-ova, CT26, and Lewis Lung. In addition, ARG inhibitor showed signs of in vivo immune cell activation including ~doubling of the number of CD8+ T cells, NK cells, and CD103+ dendritic cells in the tumor microenvironment. Combining with anti PD-L1 further increased the number of CD8+ T cells to ~4-fold of control levels. Arginase inhibitor also increased the activation state of CD8 T cell as determined by percent of granzyme, IFNg, and Ki67 positivity. Moreover, ARG inhibitor resulted in an increase of IFNg and TNFa producing CD8+ T cells in tumor draining lymph nodes. Treatment of tumor bearing mice with ARG inhibitor as monotherapy resulted in modest, but consistent, tumor growth inhibition (TGI) of ~30% in multiple syngeneic models (LL, MC38-ova, CT26). Combining ARG inhibitor with anti-PDL1 significantly improved efficacy reaching ~87% TGI in the MCA38-ova model, with 7/10 mice showing tumor regression. In summary, our pre-clinical data demonstrates that an ARG inhibitor in combination with a checkpoint inhibitor can increase plasma and tumor arginine levels and reverse tumor immune-suppression leading to strong immune activation and anti-tumor responses, suggesting arginase inhibitors could provide an opportunity to increase activity of checkpoint inhibitors clinically. Citation Format: Alwin Schuller, Aatman Doshi, Susan Cantin, Michael Secinaro, Yanjun Wang, Laura Prickett, Sharon Tentarelli, Eric Gangl, Horma Ghadially, Kristina Ilieva, Theresa Proia, Scott Mlynarski, Ray Finlay, Wenlin Shao. Inhibition of arginase in combination with anti-PDL1 leads to increased infiltration and activation of CD8+ T cells, NK cells, and CD103+ dendritic cells in mouse syngeneic tumor models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4523.

Abstract IA19: Lymphatic vessels: Barriers to adaptive immunity in melanoma

Abstract Mechanisms of immune suppression in peripheral tissues counteract protective immunity to prevent immunopathology and are co-opted by tumors for immune escape. Nonhematopoietic cells, as the resident tissue scaffold, play critical roles in regulating the infiltration, retention, function, and exit of tissue- and tumor-infiltrating leukocytes. While lymphatic vessels facilitate T-cell priming through dendritic cell and antigen delivery to lymph nodes, they also facilitate regional metastasis and exert multiple immune-suppressive mechanisms that maintain peripheral tolerance in steady state lymph nodes. We hypothesized that in tumors peripheral lymphatic vessels exhibit context-dependent function that limits local effector CD8+ T-cell accumulation in melanoma. Our work demonstrates that lymphatic vessels both maintain homeostatic mechanisms of peripheral tolerance in poorly inflamed tumors to limit immune surveillance and activate mechanisms of adaptive immune resistance in the context of potent cytotoxic T-cell activity. Consequently, we present tumor-associated lymphatic vessels as a new, anatomic immune checkpoint in melanoma. Our work suggests that understanding context-dependent lymphatic vessel function will continue to reveal novel mechanisms of immune control and metastasis that may be targeted for therapeutic response. Citation Format: Amanda W. Lund. Lymphatic vessels: Barriers to adaptive immunity in melanoma [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr IA19.

Multiple Immune-Suppressive Mechanisms in Fibrolamellar Carcinoma.

Fibrolamellar carcinoma (FLC) is a rare type of liver cancer that affects adolescents and young adults. The most effective treatment for FLC is surgical resection, but no standardized systemic therapy exists for patients with recurrent or unresectable FLC. As a first step to understand the immune microenvironment of FLC, we investigated targetable immune-checkpoint pathways, PD-1, PD-L1, B7-H3, IDO-1, and LAG3, in relation to CD8+ cytotoxic T-lymphocyte density. Thirty-two FLC tumor specimens were analyzed using IHC staining for PD-L1, CD8, PD-1, IDO, LAG3, and B7-H3. Sixty-three percent of FLC cases demonstrated membranous PD-L1 expression on tumor cells, and almost 70% of cases demonstrated PD-L1+ tumor-infiltrating lymphocytes and tumor-associated macrophages (TIL/TAM). Myeloid-derived cells appeared to be a major component of PD-L1+ tumor-infiltrating immune cells. Forty percent of the cases showed B7-H3 expression in the tumor zone, with 91% cases showing B7-H3 expression in TILs and TAMs. IDO and PD-1 expression was highest in the tumor interface zone. B7-H3 or IDO expression on tumor cells significantly correlated with higher CD8+ T-cell density. In conclusion, a high proportion of FLC cases showed robust expression of PD-1, PD-L1, B7-H3, and IDO in an adaptive immune-resistance pattern. Our findings provide further basis for targeting these different immune-checkpoint axes in FLC.

Abstract 1688: Enhancement of immune checkpoint PD-1 blockade efficacy in ovarian cancer

Abstract Introduction Advanced stage ovarian cancer is often asymptomatic and only twenty percent of patients with this diagnosis have a survival duration of five years. Immune checkpoint molecule programmed cell death-1 (PD-1) and its ligand, PD-L1, limit T cell immune responses, and thus are immunosuppressive. Clinical trials using anti-PD-1 or anti-PD-L1 antibody treatment has resulted in objective response rates (ORR) in only 10-15% of ovarian cancer patients. Our ultimate goal is to improve this outcome. Recepteur d'origine nantais (RON) is a c-Met related tyrosine kinase which binds to macrophage stimulatory protein (MSP). RON is expressed by myeloid suppressor cells and tumor cells. Inhibition of RON/ MSP ligation upregulates STAT-1 and IL-12 in macrophages, driving IFN-γ production in CD8+T cells and might potentiate the efficacy of anti-PD-1 antibody treatment. Objectives and Methods We hypothesize that combination therapy of anti-PD-1 blocking antibody which restores T cell proliferative and cytotoxic functions, combined with therapy targeting other suppressive pathways, will concomitantly overcome multiple immune suppressive mechanisms, and prevent ovarian cancer in mice. In a pilot study, ovarian cancer was induced in female C57BL/6 (H-2Kb) mice (Jackson Laboratories) at 8 weeks old, by intraperitoneal (I.P.) injection of 1 x 106 ID8-RFP ovarian tumor cells in 5 mice/ group. We determined the efficacy of anti-PD-1 antibody (RMP1-14, BioXCell; 200ug/ dose, 0.5 ml vol. I.P., 4 doses) given alone or with a RON inhibitor, BMS-777607 (each dose 50 mg/ kg body weight, 20 doses orally over 5 weeks; Selleck Chemicals). Mice were euthanized at about day 70 when control mice with disease had extended abdomens. Results Treatment of mice with an anti-PD-1 blocking antibody combined with a RON inhibitor resulted in disease improvement in 5/5 mice. The average volume of ascites recovered from 5 combination treated mice was 1.62 ml (2/5 mice had no ascites) compared with 6.64 ml from the corresponding control (a 4-fold reduction in ascites with combination treatment). There was an average 2 fold reduction in ascites volume in anti-PD-1 antibody treated mice in comparison with IgG controls (4.14 ml versus 8.62 ml). BMS-777607 treatment (vs vehicle) did not significantly alter ascites volume. Flow cytometry evaluation of spleen cells showed that in the combination treated mouse group, there was an increase in the average percentage of CD3 (combined treatment group 26.7 vs control 8.5%), CD4 (10.6 vs control 5.0%) and CD8 T cells (10.6 vs control 2.7%). We are investigating changes in immune responses genes in RNA of intestines and livers. Conclusions Anti-PD-1 antibody treatment is effective in treating ovarian cancer in mice, but treatment with this agent combined with RON/ c-Met inhibitor, BMS-777607 is superior to either single therapy. Combination treatment with these 2 agents holds promise as a novel therapeutic approach for ovarian cancer. Citation Format: Maureen Drakes, Swati Mehrotra, Ronald Potkul, Yueying Liu, M. Sharon Stack, Patrick Stiff. Enhancement of immune checkpoint PD-1 blockade efficacy 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 1688.

Antigen-Specific Antitumor Responses Induced by OX40 Agonist Are Enhanced by the IDO Inhibitor Indoximod.

Although an immune response to tumors may be generated using vaccines, so far, this approach has only shown minimal clinical success. This is attributed to the tendency of cancer to escape immune surveillance via multiple immune suppressive mechanisms. Successful cancer immunotherapy requires targeting these inhibitory mechanisms along with enhancement of antigen-specific immune responses to promote sustained tumor-specific immunity. Here, we evaluated the effect of indoximod, an inhibitor of the immunosuppressive indoleamine-(2,3)-dioxygenase (IDO) pathway, on antitumor efficacy of anti-OX40 agonist in the context of vaccine in the IDO- TC-1 tumor model. We demonstrate that although the addition of anti-OX40 to the vaccine moderately enhances therapeutic efficacy, incorporation of indoximod into this treatment leads to enhanced tumor regression and cure of established tumors in 60% of treated mice. We show that the mechanisms by which the IDO inhibitor leads to this therapeutic potency include (i) an increment of vaccine-induced tumor-infiltrating effector T cells that is facilitated by anti-OX40 and (ii) a decrease of IDO enzyme activity produced by nontumor cells within the tumor microenvironment that results in enhancement of the specificity and the functionality of vaccine-induced effector T cells. Our findings suggest a translatable strategy to enhance the overall efficacy of cancer immunotherapy. Cancer Immunol Res; 6(2); 201-8. ©2018 AACR.

Inhibition of tumor growth by cancer vaccine combined with metronomic chemotherapy and anti-PD-1 in a pre-clinical setting

Tumor microenvironment (TME) is characterized by multiple immune suppressive mechanisms able to suppress anti-tumor effector cell immunity. Combinatorial strategies, including vaccine and immunomodulatory drugs, need to be developed for improved immunotherapy efficacy.A novel combinatorial approach was assessed in C57BL/6 mice injected with mouse melanoma B16F10 cells. A multi-peptide vaccine (PEPT) was combined with a low dose metronomic chemotherapy (MCT) and an anti-PD-1 checkpoint inhibitor (CI). Statistical analysis were performed with the unpaired two-sided Student’s t-test and ANOVA.Animals treated with the multi-peptide vaccine combined with MCT or CI showed remarkable delay in tumor growth and prolonged survival as compared to control groups. The multi-pronged combination including PEPT+MCT+CI was able to prolong survival in all mice and inhibit tumor growth in 66.6% of mice. All animals which did not show tumor growth were re-challenged with the same melanoma cells and one of them showed complete tumor growth inhibition. The anti-tumor effect was associated with strong T cell immune response to vaccine mutated peptides and significant reduction of regulatory T cells.The combination of a vaccine with MCT and CI was highly efficient in potentiating the vaccine’s anti-tumor effects. The approach is highly promising to be moved into clinical trial.

Cooperative anti-tumor effect of soluble NKG2D ligand MIC neutralization and PD-1 pathway blockade

Abstract Shedding of the human NKG2D ligand MIC from tumor cell surfaces correlates with progression of many epithelial cancers. Soluble MIC enables tumor immune escape through multiple immune suppressive mechanisms, such as disturbing natural killer (NK) cell homeostatic maintenance, impairing NKG2D expression on NK cells and effector T cells, and facilitating the expansion of arginase I+ MDSCs. Our recent studies have demonstrated that sMIC is an effective cancer therapeutic target. We thus sought to investigate whether targeting sMIC using a nonblocking monoclonal antibody would enhance immunotherapies currently in use, such as PD-1 blockade therapy, using genetically engineered transplantable syngeneic sMIC+ tumor models. We demonstrate that combination therapy of a sMIC-neutralizing antibody and anti-PDL1 improved the survival of animals bearing sMIC+ tumors, in comparison to monotherapy. We further demonstrate that combination therapy augmented the activation and functional potential of NK cells, CD8 T cells, and CD4 T cells. Combined therapy significantly enhanced antigen-specific CD8 T and CD4 cell responses. Combined therapy also re-modulated the tumor microenvironment to be more immune activating and reduced neovascularization. Our findings provide a rationale for co-targeting sMIC to enhance the therapeutic efficacy of PD-1 blockade.

Abstract MS1-2: MS1-2 Breast cancer immunotherapy: Building on clinical success

Abstract Breast cancer has long been considered immunologically silent, but it is now clear that the immune system is active in this disease. Breast cancers can naturally induce tumor infiltrating lymphocytes (TIL), which represent a biomarker of the endogenous immune response. Increasing data support the association of TIL with a better prognosis for breast cancer patients, and also suggest that the presence of TIL can predict response to standard breast cancer therapies. These observations have stimulated intense interest in harnessing the natural breast cancer-specific immune response for therapeutic benefit. Breast cancer vaccines that activate the breast cancer-specific immune response have been tested for many years, with minimal clinical success to date. This is likely due to both inadequate vaccination platforms and dominant mechanisms of immune suppression active within the breast tumor microenvironment. In the tumor microenvironment, the immune checkpoint pathway governed by the programmed cell death-1 (PD-1) receptor and its ligand PD-L1 has emerged as a significant mechanism of immune resistance in a variety of cancers, including breast cancer. Several monoclonal antibody antagonists of PD-1 or PD-L1 unleash the activity of tumor-specific T cells in multiple types of cancer, resulting in durable clinical responses that translate into an overall survival benefit. Early data in breast cancer reveal that these agents have activity in some patients with triple negative and estrogen receptor-positive breast cancer, with response rates that range from about 5-44%. Antagonists of the PD-1 pathway are typically well-tolerated in breast cancer patients, with unique immune-related adverse events that are quite manageable. Selecting patients for PD-L1 expression appears to enrich for responders to single agent therapies that target PD-1/PD-L1. Based on early clinical data, agents that target the PD-1 pathway are under accelerated clinical development for breast cancer treatment. The challenge moving forward is converting immunotherapy non-responders to responders. Combination immunotherapies that integrate standard breast cancer therapies and/or multiple innovative immune-based therapies to simultaneously promote T cell activity and relieve multiple mechanisms of immune suppression at the tumor site have the greatest potential for enhancing the clinical activity of immunotherapy for breast cancer patients. Citation Format: Emens LA. MS1-2 Breast cancer immunotherapy: Building on clinical success [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr MS1-2.

Abstract PR09: Antibody-mediated blockade of phosphatidylserine synergizes with immune checkpoint blockade by inhibiting multiple immune suppressive mechanisms

Abstract Background: The expression of phosphatidylserine (PS) on cell surfaces drives immunosuppressive mechanisms associated with tolerogenic cell death. In the tumor microenvironment, PS is exposed on tumor cells and tumor vascular endothelial cells treated with conventional therapies. PS signals through multiple immune cell signaling receptors where it drives the expansion of myeloid-derived suppressor cells (MDSCs), regulatory T cells, M2 macrophages, and stimulates their production of immunosuppressive cytokines (e.g., TGFβ and IL-10), rendering the tumor microenvironment non-responsive to immune activation. PS targeting antibodies have significant anti-tumor effects in multiple tumor xenografts, syngeneic tumors, and genetically engineered preclinical tumor models through mechanisms that re-program the tumor microenvironment from immunosuppressive to immune potentiating. Methods: The combination of PS-targeting and anti-PD1 antibodies was compared to single agent therapy in multiple pre-clinical mouse models, including syngeneic melanoma and breast tumors, and a genetically engineered mouse model (GEMM) of pancreatic cancer. Mice were treated weekly IP at 5 mg/kg with a PS targeting antibody, anti-PD1, or the combination for the duration of each experiment; tumor and spleen tissue microenvironments were immune profiled by FACS, ELISPOT, and immunohistochemistry. Results: In all tumor models examined, the anti-tumor effect of combination therapy was significantly superior to single agent therapy. In B16 and K1735 melanoma tumors, the combination more than doubled the anti-tumor effect of anti-PD-1 alone. Combination therapy significantly inhibited tumor growth by over 90% in E077.1 breast tumors and prolonged animal survival by 30% (n>25 each group) in the GEMM of pancreatic cancer despite the fact that anti-PD1 therapy alone was ineffective in each of these models. Analysis of immune cell subsets in the tumor microenvironment indicated that the combination of antibody-mediated blockade of PS and PD-1 significantly enhanced the effector function of tumor infiltrating CD8+ T cells and increased the ratio of T effector to T regulatory cells, as demonstrated by significant increases in TILs producing IFNγ, TNFα, IL-2, granzyme B, and Ki-67. Combination therapy re-conditioned the tumor microenvironment to favor immune potentiation, as demonstrated by significant decreases in the frequency of MDSC, the ratio of M2 to M1 macrophages, the expression of surface PD-L1, and reductions in TGFβ and IL-10. Furthermore, combination treatment induced systemic tumor specific CD8 T cell immunity, as mice demonstrating complete responses (in the K1735 and E077.1 models) rejected tumor cells upon re-challenge. Splenocytes from these mice had significantly higher numbers of tumor-specific IFNγ-producing cells in ELISPOT assay. Studies were completed without toxicity in any setting. Conclusions: Antibody-mediated PS blockade inhibits PS-mediated immune suppression and stimulates FcRγ activation. This results in immune activation that enables an otherwise non-responsive tumor microenvironment to respond to checkpoint inhibition. This abstract is also presented as Poster B08. Citation Format: Xianming Huang, Jian Gong, Michael Gray, Van Nguyen, Ryan Parks, Chris Hughes, Jeff Hutchins, Rolf Brekken, Bruce Freimark. Antibody-mediated blockade of phosphatidylserine synergizes with immune checkpoint blockade by inhibiting multiple immune suppressive mechanisms. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr PR09.

Cooperative therapeutic anti-tumor effect of IL-15 agonist ALT-803 and co-targeting soluble NKG2D ligand sMIC.

Shedding of the human NKG2D ligand MIC (MHC class I-chain-related molecule) from tumor cell surfaces correlates with progression of many epithelial cancers. Shedding-derived soluble MIC (sMIC) enables tumor immune escape through multiple immune suppressive mechanisms, such as disturbing natural killer (NK) cell homeostatic maintenance, impairing NKG2D expression on NK cells and effector T cells, and facilitating the expansion of arginase I+ myeloid suppressor cells. Our recent study has demonstrated that sMIC is an effective cancer therapeutic target. Whether targeting tumor-derived sMIC would enhance current active immunotherapy is not known. Here, we determined the in vivo therapeutic effect of an antibody co-targeting sMIC with the immunostimulatory IL-15 superagonist complex, ALT-803, using genetically engineered transplantable syngeneic sMIC+ tumor models. We demonstrate that combined therapy of a nonblocking antibody neutralizing sMIC and ALT-803 improved the survival of animals bearing sMIC+ tumors in comparison to monotherapy. We further demonstrate that the enhanced therapeutic effect with combined therapy is through concurrent augmentation of NK and CD8 T cell anti-tumor responses. In particular, expression of activation-induced surface molecules and increased functional potential by cytokine secretion are improved greatly by the administration of combined therapy. Depletion of NK cells abolished the cooperative therapeutic effect. Our findings suggest that administration of the sMIC-neutralizing antibody can enhance the anti-tumor effects of ALT-803. With ALT-803 currently in clinical trials to treat progressive solid tumors, the majority of which are sMIC+, our findings provide a rationale for co-targeting sMIC to enhance the therapeutic efficacy of ALT-803 or other IL-15 agonists.

T Cells Engineered against a Native Antigen Can Surmount Immunologic and Physical Barriers to Treat Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinomas (PDAs) erect physical barriers to chemotherapy and induce multiple mechanisms of immune suppression, creating a sanctuary for unimpeded growth. We tested the ability of T cells engineered to express an affinity-enhanced T cell receptor (TCR) against a native antigen to overcome these barriers in a genetically engineered model of autochthonous PDA. Engineered T cells preferentially accumulate in PDA and induce tumor cell death and stromal remodeling. However, tumor-infiltrating T cells become progressively dysfunctional, a limitation successfully overcome by serial T cell infusions that resulted in a near-doubling of survival without overt toxicities. Similarly engineered human T cells lyse PDA cells in vitro, further supporting clinical advancement of this TCR-based strategy for the treatment of PDA.

Abstract 3364: Epithelial-mesenchymal transition is associated with a profound inflammatory tumor microenvironment in lung adenocarcinoma

Abstract Promising results in the treatment of non-small cell lung cancer (NSCLC) have been seen with immunomodulatory agents targeting immune checkpoints, such as programmed cell death 1 (PD-1) or programmed cell death 1 ligand (PD-L1). However, only a select group of patients respond to these interventions. The identification of biomarkers that predict clinical benefit to immune-based approaches, such as immune checkpoint blockade, is critical to successful translation of these agents. By using integrated gene array analysis in three large independent NSCLC patient datasets along with immunohistochemistry study (IHC), reverse phase protein array (RPPA) and in vivo animal study, we demonstrated that epithelial-mesenchymal transition (EMT) is highly associated with a profound inflammatory tumor microenvironment in lung adenocarcinoma. Our data revealed immune activation and simultaneous development of multiple immune suppressive mechanisms, including elevation of immune checkpoints such as PD-L1, PD-L2, PD-1, TIM-3, BTLA and CTLA-4, as well as an increase in tumor infiltrating CD4+Foxp3+ regulatory T cells, IL-6 and indoleamine 2,3-dioxygenase (IDO) in lung adenocarcinomas with a mesenchymal phenotype. Our data suggested that EMT might represent a potential biomarker to select the patients who will benefit from immune checkpoint blockade agents and other immunotherapies in NSCLC and possibly other cancers. Note: This abstract was not presented at the meeting. Citation Format: Yanyan Lou, Lixia Diao, Parra Cuentas Edwin Roger, Warren L. Denning, Limo Chen, Youhong Fan, Jaime Rodriguez, Lauren Byers, Jing Wang, Vassiliki Papadimitrakopoulou, Behrens Carmen, Ignacio I. Wistuba, Patrick Hwu, John V. Heymach, Don L. Gibbons. Epithelial-mesenchymal transition is associated with a profound inflammatory tumor microenvironment in lung adenocarcinoma. [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 3364. doi:10.1158/1538-7445.AM2015-3364

BRAFV600E Co-opts a Conserved MHC Class I Internalization Pathway to Diminish Antigen Presentation and CD8+ T-cell Recognition of Melanoma.

Oncogene activation in tumor cells induces broad and complex cellular changes that contribute significantly to disease initiation and progression. In melanoma, oncogenic BRAF(V600E) has been shown to drive the transcription of a specific gene signature that can promote multiple mechanisms of immune suppression within the tumor microenvironment. We show here that BRAF(V600E) also induces rapid internalization of MHC class I (MHC-I) from the melanoma cell surface and its intracellular sequestration within endolysosomal compartments. Importantly, MAPK inhibitor treatment quickly restored MHC-I surface expression in tumor cells, thereby enhancing melanoma antigen-specific T-cell recognition and effector function. MAPK pathway-driven relocalization of HLA-A*0201 required a highly conserved cytoplasmic serine phosphorylation site previously implicated in rapid MHC-I internalization and recycling by activated immune cells. Collectively, these data suggest that oncogenic activation of BRAF allows tumor cells to co-opt an evolutionarily conserved MHC-I trafficking pathway as a strategy to facilitate immune evasion. This link between MAPK pathway activation and the MHC-I cytoplasmic tail has direct implications for immunologic recognition of tumor cells and provides further evidence to support testing therapeutic strategies combining MAPK pathway inhibition with immunotherapies in the clinical setting.

Immune combinational therapy targeting OX40 and IDO synergistically enhances efficacy of a cancer vaccine

Enhancing an effector immune response by cancer vaccines has not been clinically successful so far. Although the necessary immune response may be elicited using vaccine-based therapies, this has not been sufficient for a positive clinical outcome; since most cancers can escape immune surveillance via multiple immune suppressive mechanisms induced in the tumor environment. A relatively recent strategy to increase the therapeutic efficacy of tumor vaccination is to combine it with alternative immunotherapeutic approaches. Here we tested if simultaneous targeting of the effector arm of the immune system with an agonist anti-OX40 mAb together with targeting of suppressive mechanisms in the tumor, via inhibition of indoleamine-(2,3)-dioxygenase (IDO), could enhance the anti-tumor activity of vaccine in mouse models of cancer. The OX40 molecule is a co-stimulatory receptor expressed on T-cells that can lead to proliferation and enhancement of T-cell effector function when triggered with an agonistic antibody. However, the tumor secretes a number of suppressive signals as a protective mechanism against its destruction, one of which is IDO. We demonstrate that treatment of TC-1 tumor bearing mice with an anti-OX40 agonist antibody in combination with vaccine leads to the enhancement of antigen-specific T cell responses. The dose of 1 mg/kg anti-OX40 antibody stimulated the effector arm of T cells, which ultimately led to a significant increase in the CD8+/regulatory T-cell (Treg) ratio within tumors. Further, this combination of vaccine and anti-OX40 antibody treatment led to a significant inhibition of tumor growth and to a prolonged survival of tumor bearing mice compared to control, and resulted in complete tumor regression in 20% of treated mice. We found, however, that this anti-tumor activity could be further enhanced through the combination of vaccine, anti-OX40 and 1-methyltryptophan (1-MT), an IDO activity inhibitor. IDO has been shown to be secreted by tumor cells, suppressive dendritic cells and macrophages in tumor environment, and is known to be responsible for suppressing effector cells and inducing Tregs. Our data demonstrate that vaccine/anti-OX40 antibody/1-MT combination leads to a more profound inhibition of tumor growth in mice and complete regression of established tumors in 60% of treated mice. In conclusion, we provide evidence that simultaneous targeting of the effector arm of the immune system, via anti-OX40 antibody, and suppressive mechanisms within the tumor, via 1-MT, in combination with cancer vaccine has a synergistic effect for tumor eradication and is a promising strategy that could potentially enhance the overall efficacy of cancer treatment in patients.

T Regulatory Cells and Helicobacter pylori-Associated Diseases

In recent years, a crucial role for T regulatory cells (Tregs) in the pathophysiology of infectious, neoplastic and immune-mediated gastrointestinal diseases has been described. These cells represent a continuously dynamic and diverse population, mainly comprised naturally occurring and inducible Tregs. Multiple mechanisms of immune suppression are utilized by Tregs in order to regulate virtually all immune cells. These peculiar characteristics are being increasingly recognized as important determinants in gut-associated homeostasis and autoimmunity and, in particular, in Helicobacter pylori (H. pylori)-associated diseases. In this regard, Tregs have been implicated not only in the pathogenesis of the immune sequelae of this chronic infection, but also in H. pylori-related malignancies. This review attempts to present a comprehensive approach to Tregs biology and function for preserving homeostasis. In addition, current evidence for their contribution mainly to inflammatory and immune-mediated H. pylori-associated diseases is critically reviewed.

Tumour eradication and induction of memory against murine mesothelioma by combined immunotherapy

Numerous immunotherapy treatments for cancer are undergoing clinical trials or are already approved for use. One particular area of interest is targeting mechanisms of immune tolerance. Using a murine model of mesothelioma, we investigated the roles of regulatory T-cells, intratumoural transforming growth factor (TGF)-β and the negative regulator molecule cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) in immune tolerance to tumours. It was found that treatments targeting a single negative regulator molecule mechanism were not as effective against tumours as targeting multiple mechanisms simultaneously. Most importantly, it was found that a combined triple treatment of anti-CD25 monoclonal antibody (mAb), anti-CTLA-4 mAb and TGF-β soluble receptor resulted in long-term clearance of tumours and memory against tumour rechallenge. These data suggest that clinical application of immunotherapies against tumours may be improved by simultaneously targeting multiple mechanisms of immune suppression.

Multiple mechanisms of immune suppression by B lymphocytes.

Suppression of the immune system after the resolution of infection or inflammation is an important process that limits immune-mediated pathogenesis and autoimmunity. Several mechanisms of immune suppression have received a great deal of attention in the past three decades. These include mechanisms related to suppressive cytokines, interleukin (IL)-10 and transforming growth factor (TGF)-β, produced by regulatory cells, and mechanisms related to apoptosis mediated by death ligands, Fas ligand (FasL) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), expressed by killer or cytotoxic cells. Despite many lines of evidence supporting an important role for B lymphocytes as both regulatory and killer cells in many inflammatory settings, relatively little attention has been given to understanding the biology of these cells, their relative importance or their usefulness as therapeutic targets. This review is intended to give an overview of the major mechanisms of immunosuppression used by B lymphocytes during both normal and inflammatory contexts. The more recent discoveries of expression of granzyme B, programmed death 1 ligand 2 (PD-L2) and regulatory antibody production by B cells as well as the interactions of regulatory and killer B cells with regulatory T cells, natural killer T (NKT) cells and other cell populations are discussed. In addition, new evidence on the basis of independent characterizations of regulatory and killer CD5(+) B cells point toward the concept of a multipotent suppressor B cell with seemingly high therapeutic potential.

Control of intestinal inflammation by regulatory T cells.

Transfer of CD4+ T cells to immune-deficient mice in the absence of the CD25+ subset leads to the development of colitis, indicating that regulatory cells capable of controlling a bacteria-driven inflammatory response are present in normal mice. Cells with this function are present in the thymus as well as in the periphery of germ-free mice, suggesting they may be reactive with self-antigen. These cells resemble CD4+CD25+ cells that inhibit organ-specific autoimmunity, suggesting that a similar subset of regulatory T cells may control responses to self and foreign antigens. Development of colitis is dependent on accumulation of activated CD134L+ dendritic cells (DC) in the mesenteric lymph nodes, which is inhibited by CD4+CD25+ cells, indicating that regulatory T cells may control DC activation in vivo. Whilst inhibition of T-cell activation in vitro by CD4+CD25+ cells does not involve interleukin-10 and transforming growth factor-beta, these cytokines are required for the suppression of colitis. It may be that control of responses that activate the innate immune system requires multiple mechanisms of immune suppression. Recently, we identified CD4+CD25+ cells with immune suppressive activity in the thymus and peripheral blood of humans, raising the possibility that dysfunction in this mechanism of immune regulation may be involved in the development of autoimmune and inflammatory diseases.