Glucocorticoid-induced Tumor Necrosis Factor Receptor Research Articles

Enhanced immunotherapy by combining a vaccine with a novel murine GITR ligand fusion protein

Immunotherapy was significantly enhanced in a murine tumor model by combining a vaccine with a fusion protein designed to target the glucocorticoid-induced tumor necrosis factor (TNF) receptor related gene (GITR) on the surface of T cells. The recombinant poxvirus-based vaccine platform included Modified Vaccinia virus Ankara (rMVA) and fowlpox (rF) vectors as the driver immunogens both engineered to express the human carcinoembryonic antigen (CEA) and three murine costimulatory molecules B7.1, ICAM-1, LFA-3 (designated TRICOM). In previous studies, mice expressing human CEA as a transgene (CEA.Tg mice) vaccinated with rMVA/rF-CEA-TRICOM overcame CEA immune tolerance by inducing anti-CEA‒specific immunity and regression of CEA-expressing tumors. The murine GITR ligand fusion protein (mGITRL-FP) consisted of a mouse IgG2a Fc region, a yeast-derived coiled GCN4 pII and the extracellular GITR-binding domain of murine GITR ligand. The design maximized valency and the potential to agonize the GITR receptor. Combined treatment of the vaccine and mGITRL-FP mediated a more robust tumor regression, leading to sustained improvement in overall survival. The enhanced immunotherapeutic effect was linked to the generation of a strong CD8+ T cell antitumor immune response. A treatment schedule with mGITRL-FP administered prior to the priming rMVA-CEA-TRICOM vaccination was of paramount importance. The mechanism of action for the enhanced antitumor effects resided in the depletion of immune cells, particularly FoxP3+ regulatory T cells, that express high GITR levels following activation. The results provide evidence that targeting GITR with mGITRL-FP in concert with a cancer vaccine represents a potential novel approach to more effective immunotherapy.

Abstract 5621: Prevalence of GITR expression and pharmacodynamic (PD) biomarkers in syngeneic tumor models treated by a GITR agonist (GITRL-Fc)

Abstract GITRL (Glucocorticoid-Induced Tumor Necrosis Factor Receptor Ligand, TNFSF18) is a member of the tumor necrosis factor (TNF) ligand superfamily. GITRL binds and activates the co-stimulatory surface receptor GITR, which promotes proliferation and activation of effector T cells (Teff) and inhibits suppressive activity of regulatory T cells (Treg). It is thus hypothesized that co-stimulation of GITR by agonist agents will promote anti-tumor immunity. We generated a novel single-gene GITRL trimer fused to an immunoglobulin Fc domain (GITRL-Fc) that shows robust single agent antitumor efficacy and immune effects in multiple syngeneic mouse models, suggesting its potential benefit in cancer immunotherapy To investigate the prevalence of GITR expression in human tumors, RNA-Seq data analyses of 33 tumor types in TCGA showed GITR is highly expressed in a subset of solid tumors, including head & neck, lung, breast, esophageal, and bladder cancers. In most solid tumors, GITR expression correlated poorly with T cell markers, implying that GITR may not be exclusive to immune cells and may be expressed in tumor cells as well. Similar findings emerged from RNA-Seq data analysis of patient-derived xenograft (PDX) samples from 24 tumor types. The gene expression data was corroborated by immunohistochemistry (IHC) analysis of GITR expression in 17 tumor types which showed that in addition to immune cells, GITR was expressed on tumor cell membranes. A multi-platform approach was taken to investigate GITRL-Fc pharmacodynamic (PD) biomarkers in tumors and in matched whole blood samples from mice bearing CT26 colon, 4T1 breast, and B16F10 melanoma carcinoma models. Global gene expression levels were profiled by microarray on treated and control tissues. We also monitored the changes of immune cell populations and cytokine secretions by flow cytometry, Luminex and IHC. Immune gene changes were more robust in tumors than in blood samples. In tumor samples, GITRL-Fc increased the gene expression associated with T cells, CD8 T cells, cytotoxicity, Th1 cells, interferon gamma (IFN-γ), natural killer cells, Teff cells, and T cell activation markers. These gene changes were validated by quantitative real-time PCR. Similarly, flow cytometry analysis showed that GITRL-Fc promoted activation of CD4+ effector cells, decreased Treg frequency, and increased the ratio of CD8+ T cell/Treg in the tumor. GITRL-Fc also modulated secretion of cytokines in splenocytes, including an increase in IFN-γ. Taken together, the PD biomarker changes in immune-related gene expression, immune cell populations, and cytokine secretions observed in these preclinical tumor models are consistent with GITRL-Fc mechanism of action and demonstrated target engagement of GITRL-Fc. Additional approaches, including in-silico sorting, to monitor rare immune cell populations in tumor samples will be discussed. Citation Format: Min Wang, Fiore Cattaruzza, Pete Yeung, Alayne Brunner, Erwan LeScolan, Yuwang Liu, Jennifer Cain, Gilbert OYoung, Earth Light Lowe, Belinda Cancilla, Rose Harris, Tim Hoey, Austin Gurney, John Lewicki, Gretchen Argast, Ann M. Kapoun. Prevalence of GITR expression and pharmacodynamic (PD) biomarkers in syngeneic tumor models treated by a GITR agonist (GITRL-Fc) [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 5621. doi:10.1158/1538-7445.AM2017-5621

GITR ligand fusion protein agonist enhances the tumor antigen\u2013specific CD8 T-cell response and leads to long-lasting memory

BackgroundThe expansion of antigen-specific CD8 T cells is important in generating an effective and long-lasting immune response to tumors and viruses. Glucocorticoid-induced tumor necrosis factor receptor family-related receptor (GITR) is a co-stimulatory receptor that binds the GITR ligand (GITRL). Agonism of GITR can produce important signals that drive expansion of effector T cell populations.MethodsWe explored two separate murine tumor models, CT26 and TC-1, for responsiveness to GITR Ligand Fusion Protein(GITRL-FP) monotherapy. In TC-1, GITRL-FP was also combined with concurrent administration of an E7-SLP vaccine. We evaluated tumor growth inhibition by tumor volume measurements as well as changes in CD8 T cell populations and function including cytokine production using flow cytometry. Additionally, we interrogated how these therapies resulted in tumor antigen-specific responses using MHC-I dextramer staining and antigen-specific restimulations.ResultsIn this study, we demonstrate that a GITR ligand fusion protein (GITRL-FP) is an effective modulator of antigen-specific CD8 T cells. In a CT26 mouse tumor model, GITRL-FP promoted expansion of antigen-specific T cells, depletion of regulatory T cells (Tregs), and generation of long-lasting CD8 T cell memory. This memory expansion was dependent on the dose of GITRL-FP and resulted in complete tumor clearance and protection from tumor rechallenge. In contrast, in TC-1 tumor–bearing mice, GITRL-FP monotherapy could not prime an antigen-specific CD8 T cell response and was unable to deplete Tregs. However, when combined with a vaccine targeting E7, treatment with GITRL-FP resulted in an augmentation of the vaccine-induced antigen-specific CD8 T cells, the depletion of Tregs, and a potent antitumor immune response. In both model systems, GITR levels on antigen-specific CD8 T cells were higher than on all other CD8 T cells, and GITRL-FP interacted directly with primed antigen-specific CD8 T cells.ConclusionsWhen taken together, our results demonstrate that the delivery of GITRL-FP as a therapeutic can promote anti-tumor responses in the presence of tumor-specific CD8 T cells. These findings support further study into combination partners for GITRL-FP that may augment CD8 T-cell priming as well as provide hypotheses that can be tested in human clinical trials exploring GITR agonists including GITRL-FP.

Therapeutic immune monitoring of CD4+CD25+ T cells in chronic myeloid leukemia patients treated with tyrosine kinase inhibitors.

Tyrosine kinase inhibitors (TKIs), including imatinib, dasatinib and nilotinib, are effective forms of therapy for various types of solid cancers and Philadelphia chromosome-positive (Ph+) chronic myeloid leukemia. A number of TKIs have been known to have strong effects on T cells, particularly cluster of differentiation (CD) 4+CD25+ T cells, also known as regulatory T cells (Tregs). There is currently a deficit in the available clinical data regarding this area of study. In the present study, a total of 108 peripheral blood samples were collected from patients with chronic myeloid leukemia (CML) at diagnosis (n=31), and at 3 and 6 months following treatment with TKI [imatinib (n=12), dasatinib (n=11) and nilotinib groups (n=8)] and healthy controls (n=15). Peripheral blood mononuclear cells were collected from the patients prior to and following TKI treatment. The subtype and number of T lymphocytes in patients and healthy donors were analyzed using flow cytometry. Additionally, flow cytometry and ELISA were used to detect the proliferation and suppression of Tregs. Expression of cytokines and other molecules [forkhead box P3 (FOXP3), glucocorticoid-induced tumor necrosis factor receptor (GITR) and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4)] were also analyzed at 3 and 6 months following treatment with TKIs. It was indicated that, at diagnosis, a similar number of lymphocytes were detected in patients and control. However, following treatment with a TKI, the number of total T cells, Tregs, CD4+ T and CD8+ T cells decreased to various degrees in patients. Furthermore, the decrease in the number of Tregs was more significant with time. Although treatment with imatinib, dasatinib and nilotinib demonstrated similar inhibitory effects on the quantity of Tregs in vivo, the TKIs exhibited differential effects on the function of Tregs in vitro. Proliferation, suppression and expression of cytokines [interleukin (IL)-4, IL-10 and transforming growth factor (TGF)-β] and molecules (FOXP3, GITR and CTLA-4) decreased significantly in treatment groups with imatinib and dasatinib. The decrease was not significant in the nilotinib treatment group. Imatinib and dasatinib may exert more marked inhibitory roles compared with nilotinib on regulating the number and function of Tregs. These results suggest that personalized treatment and follow-up of CML patients during TKI treatment, particularly for those who received post-transplant TKI treatment may be beneficial.

Murinization and H Chain Isotype Matching of the Anti-GITR Antibody DTA-1 Reduces Immunogenicity-Mediated Anaphylaxis in C57BL/6 Mice.

Recent advances in immuno-oncology have shown that the immune system can be activated to induce long-term, durable antitumor responses. For immuno-oncology drug development, immune activation is often explored using rat Abs in immunocompetent mouse models. Although these models can be used to show efficacy, antidrug immune responses to experimental protein-based therapeutics can arise. Immunogenicity of surrogate Abs may therefore represent an important obstacle to the evaluation of the antitumor efficacy of immunomodulator Abs in syngeneic models. A recent publication has shown that anti-glucocorticoid-induced TNFR family-related protein agonistic Ab DTA-1 (rat or murinized IgG2a) can induce the development of anaphylaxis in C57BL/6 mice upon repeated i.p. dosing because of an anti-idiotypic anti-drug Ab immune response. This study was undertaken to address the impact of the immunogenicity derived from the Fc and variable domains. To this end, chimerized (rat V domains/mouse constant regions) and murinized (95% mouse sequence) DTA-1-based surrogate Abs with a murine IgG2c H chain isotype were created. Chimerization and murinization of DTA-1 did not affect receptor binding and glucocorticoid-induced TNFR family-related protein-induced T cell agonistic properties. Similar in vivo antitumor efficacy and intratumoral CD8+/regulatory T cells were also observed. Finally, treatment of C57BL/6 mice with the chimerized and murinized DTA-1 Abs on a C57BL/6-matched IgG2c isotype resulted in reduced development and severity of anaphylaxis as measured by decline of body temperature, behavioral effects, serum IL-4, IgE, and anti-drug Ab levels. These results suggest that careful murinization and selection of a strain-matched H chain isotype are critical to generate ideal surrogate Abs for testing immuno-oncology mechanisms in vivo.

Preliminary results of a phase I/IIa study of BMS-986156 (glucocorticoid-induced tumor necrosis factor receptor–related gene [GITR] agonist), alone and in combination with nivolumab in pts with advanced solid tumors.

104 Background: BMS-986156 is a fully human IgG1 agonist mAb that binds GITR and promotes T effector cell activation and possible reduction/inactivation of T regulatory cells. Preclinical data show enhanced antitumor T-cell activity with anti-GITR + anti–programmed death-1 (PD-1). Here we describe preliminary dose escalation data from a phase I/IIa study of BMS-986156 ± nivolumab (anti–PD-1 mAb) in pts with advanced solid tumors (NCT02598960). Methods: During dose escalation, pts received BMS-986156 (10–800 mg) or BMS-986156 (30–800 mg) + nivolumab (240 mg) every 2 weeks. Objectives included safety (primary), immunogenicity, pharmacokinetics (PK), pharmacodynamics (PD), and efficacy. Results: As of Dec 12, 2016, 66 pts were treated with BMS-986156 (n = 29) or BMS-986156 + nivolumab (n = 37).No dose-limiting toxicities (DLTs) were reported during dose escalation. The most common treatment-related adverse events reported with BMS-986156/BMS-986156 + nivolumab included pyrexia (21%/30%), chills (10%/16%), and fatigue (14%/14%); events were G1/2 in all pts except for 4 pts (6%) treated with the combination (G3 lipase [n = 1], G3 lung infection [n = 1], G3 fatigue [n = 1], and G3 aspartate aminotransferase with G4 creatine phosphokinase [n = 1; leading to discontinuation of treatment]). Preliminary data indicate that the incidence of immunogenicity to BMS-986156 was low when BMS-986156 ± nivolumab was administered. Preliminary data also indicate that BMS-986156 ± nivolumab exhibits linear PK with dose proportionality after a single dose, and BMS-986156 ± nivolumab is biologically active in PD analyses in peripheral blood. Initial antitumor activity has been observed in several pts treated with the combination; these data will be reported. Conclusions: This is the first report of clinical data with an anti-GITR mAb ± a PD-1 inhibitor.BMS-986156 ± nivolumab was well tolerated, with no DLTs and low immunogenicity. Antitumor activity was observed with BMS-986156 + nivolumab at doses predicted to be biologically active. Further evaluation of this combination in pts with advanced solid tumors is ongoing. Clinical trial information: NCT02598960.

Expansion of CD25-Negative Forkhead Box P3-Positive T Cells during HIV and Mycobacterium tuberculosis Infection.

Tuberculosis (TB) and HIV alter the immune system, and coinfected (HIV-TB) individuals usually present deregulations of T-lymphocytic immune response. We previously observed an increased frequency of “unconventional” CD4+CD25−FoxP3+ Treg (uTreg) population during HIV-TB disease. Therefore, we aimed to explore the phenotype and function of uTreg and conventional CD4+CD25+FoxP3+ Treg subsets (cTreg) in this context. We evaluated the expression of CD39, programmed cell death protein 1 (PD1), glucocorticoid-induced tumor necrosis factor receptor (GITR), and the effector/memory distribution by flow cytometry in cTreg and uTreg. Also, IL-10, TGF-β, IFN-γ production, and the suppressor capacity of uTregs were analyzed in cocultures with effector lymphocytes and compared with the effect of regulatory T cells (Tregs). We found diminished expression of CD39 and higher levels of PD1 on uTreg compared to cTreg in both HIV-TB and healthy donors (HD). In addition, uTreg and cTreg showed differences in maturation status in both HIV-TB and HD groups, due to the expansion of effector memory uTregs. Interestingly, both HIV-TB and HD showed a pronounced production of IFN-γ in uTreg population, though no significant differences were observed for IL-10 and TGF-β production between uTreg and cTreg. Moreover, IFN-γ+ cells were restricted to the CD39− uTreg population. Finally, when the suppressor capacity was evaluated, both uTreg and cTreg inhibited polyclonal T cell-proliferation and IFN-γ production in a similar extent. These findings suggest that uTregs, which are expanded during HIV-TB coinfection, exert regulatory functions in a similar way to cTregs despite an altered surface expression of Treg characteristic markers and differences in cytokine production.

Abstract 561: GITR Activation Promotes Regulatory CD4+ T-cell Responses and Stimulates Leukocyte Recruitment in Atherosclerotic Plaques

Glucocorticoid-induced tumor necrosis factor receptor family-related protein (GITR) - a costimulatory molecule - is expressed on CD4(+) effector memory T cells and regulatory T cells as well as antigen-presenting cells and mast cells; while its ligand (GITRL) is mainly found on antigen-presenting cells and endothelial cells. However, the definitive role of GITR in atherosclerosis is not fully understood. Our hypothesis is that signaling through GITR plays a vital role in atherosclerosis progression. Low-density lipoprotein receptor-deficient mice (Ldlr -/- ) with B-cell-restricted overexpression of GITRL ( Gitrl tg ) fed a high-cholesterol diet showed a profound increase in both CD4(+) effector memory T cells and regulatory T cells in secondary lymphoid organs in comparison to wild-type controls. Additionally, the number of regulatory T cells was significantly enhanced in the thymus and aorta of these mice along with increased GITRL and interleukin-2 transcript levels. Atherosclerotic lesions of Ldlr -/- Gitrl tg mice contained more total CD3 + T cells as well as Foxp3 + regulatory T cells overall, leading to significantly less severe atherosclerosis. Conversely, atherosclerosis was found to be less severe in mice deficient in apolipoprotein E and GITR (ApoE -/- GITR -/- ). Atherosclerotic lesions in these mice were found to contain less macrophages and CD3-positive T-cells. Perfusion assays using two-photon excitation microscopy revealed less wild type leukocyte adhesion on GITR-deficient endothelium, with a further reduction in adhesion by GITR-deficient leukocytes to both wild type and GITR-deficient endothelia. Finally, expression of GITR expression in human plaque tissue was significantly increased in ruptured plaques. In conclusion, these data indicate that continuous GITR stimulation through B cell GITRL acts protective in a mouse model of atherosclerosis by regulating the balance between regulatory and effector memory CD4(+) T cells, while GITR activation on endothelial cells promotes atherogenesis by stimulating leukocyte recruitment into the plaque.

Simultaneously increased expression of glucocorticoid‑induced tumor necrosis factor receptor and its ligand contributes to increased interleukin‑5/13‑producing group 2 innate lymphocytes in murine asthma.

Glucocorticoid‑induced tumor necrosis factor receptor (GITR) is expressed at high levels on CD4+CD25+ regulatory T cells (Tregs). Following activation by its ligand (GITRL), GITR influences the activity of effector T cells and Tregs and participates in the development of numerous autoimmune and inflammatory diseases, including asthma. However, the GITR/GITRL expression level in lung tissue and its influence on group 2 innate lymphocytes (ILC2s) in asthma remains unclear. The present study detected the number of ILC2s and the expression levels of GITR and GITRL in the lung tissues of asthmatic mice by flow cytometry analysis, immunofluorescence staining and reverse transcription quantitative polymerase chain reaction. The results demonstrated that the number of ILC2s and the expression levels of ILC2‑associated molecules (interleukin‑33 receptor ST2, RAR related orphan receptor A and inducible T cell costimulator) were increased in the lung tissues of asthmatic mice. The upregulated ILC2s were accompanied by an increased number of GITR‑positive cells in the spleen and lung tissues, and additionally an increased level of GITRL mRNA in lung tissue in asthma. In addition, increased mRNA expression levels of interleukin (IL)‑5 and IL‑13 were observed in the asthmatic lung, and there was a significant, positive correlation between the mRNA levels of GITR/GITRL and ILC2‑associated molecules. Therefore, GITRL treatment may increase the number of ILC2s and/or GITR‑positive cells in lung tissue. These results indicated that the activity of GITR‑expressing ILC2s may be enhanced via interaction of GITRL and GITR, which may contribute to pathogenesis of asthma. These findings present potential therapeutic targets for the treatment of asthma.

Novel Foxp3(-) IL-10(-) Regulatory T-cells Induced by B-Cells Alleviate Intestinal Inflammation in Vivo.

Recent studies have revealed various Foxp3− regulatory T (Treg) cell subsets effectively protect mice from colitis. In the present study, we demonstrated that B cells induced a particular subset of regulatory T (Treg-of-B) cells, expressing programmed cell death 1 (PD-1), inducible costimulator (ICOS), lymphocyte-activation gene 3 (LAG3), glucocorticoid-induced tumor necrosis factor receptor (GITR), and OX-40, did not express Foxp3. Treg-of-B cells produced abundant levels of IL-10 and low levels of IL-4 and TGF-β. Adoptive transfer of Treg-of-B cells protected mice from CD4+CD45RBhi T-cell-induced colitis, including infiltration of leukocytes, depletion of goblet cells, epithelial hyperplasia, and inhibition of Th1 and Th17 cytokines. These features were similar to IL-10-producing type 1 regulatory T (Tr1) cells; however, IL-10-deficient Treg-of-B cells maintained their suppressive function in vitro as well as in vivo, while the regulation of Tr1 cells depended on IL-10. In conclusion, Treg-of-B cells protected against experimental colitis through an IL-10-independent mechanism. We reported a novel subpopulation of regulatory T cells was different from conventional Foxp3+ Treg and IL-10-producing Tr1 cells.

The changes of regulatory B cell subset in children with type 1 diabetes mellilus

The proportions of CD19+ CD24hiCD38hi regulatory B cells(Breg)and CD4+ CD25+ Treg in peripheral blood of children with type 1 diabetes mellitus(T1DM)were detected by flow cytometric analysis. The concentration of interleukin 10(IL-10)protein was measured by ELISA. The mRNA expression levels of Foxp3, cytotoxic T lymphocyte-associated antigen-4(CTLA-4), and glucocorticoid-induced tumor necrosis factor receptor(GITR)in CD4+ T cells and IL-10 in CD19+ B cells were evaluated using real-time PCR. The results showed that the proportions of CD19+ CD24hiCD38hiBreg in peripheral blood, the mRNA and protein levels of IL-10 in B cells from patients with T1DM were lower than those from healthy controls(P<0.05). The mRNA expression levels of Treg associated factors such as Foxp3, CTLA-4, and GITR were lower in CD4+ T cells from children with T1DM compared with the controls(P<0.05). These results suggest that Breg cell deficiency and dysfunction might be one of the important factors causing cellular immune dysfunction in patients with T1DM. (Chin J Endocrinol Metab, 2016, 32: 584-586) Key words: Diabetes mellitus, type 1; Regulatory B cells; Regulatory T cells; Interluekin-10

Constitutive GITR Activation Reduces Atherosclerosis by Promoting Regulatory CD4 + T-Cell Responses—Brief Report

Glucocorticoid-induced tumor necrosis factor receptor family-related protein (GITR) is expressed on CD4(+) effector memory T cells and regulatory T cells; however, its role on these functionally opposing cell types in atherosclerosis is not fully understood. Low-density lipoprotein receptor-deficient mice (Ldlr(-/-)) were lethally irradiated and reconstituted with either bone marrow from B-cell-restricted Gitrl transgenic mice or from wild-type controls and fed a high-cholesterol diet for 11 weeks. Chimeric Ldlr(-/-) Gitrl(tg) mice showed a profound increase in both CD4(+) effector memory T cells and regulatory T cells in secondary lymphoid organs. Additionally, the number of regulatory T cells was significantly enhanced in the thymus and aorta of these mice along with increased Gitrl and Il-2 transcript levels. Atherosclerotic lesions of Ldlr(-/-) Gitrl(tg) chimeras contained more total CD3(+) T cells as well as Foxp3(+) regulatory T cells overall, leading to significantly less severe atherosclerosis. These data indicate that continuous GITR stimulation through B cell Gitrl acts protective in a mouse model of atherosclerosis by regulating the balance between regulatory and effector memory CD4(+) T cells.

Abstract 3215: GITRL-Fc can significantly reduce tumor growth by stimulating innate and adaptive immunity

Abstract Although the interaction between GITR (Glucocorticoid-Induced Tumor Necrosis Factor Receptor) and GITRL (its ligand) is important for the development of immune responses, the cellular mechanisms underlying anti-tumor immunity including both innate and adaptive immunity is not fully understood. We generated a novel single-gene linkerless GITRL trimer fused to an immunoglobulin Fc domain (GITRL-Fc), and investigated its effect on controlling tumor growth and immune responses in preclinical tumor models. Treatment with GITRL-Fc significantly reduced tumor growth in several preclinical tumor models by inducing Th1 biased anti-tumor immunity and reducing Treg-mediated immune suppression. Immune cell depletion studies showed that anti-tumor immunity induced by GITRL-Fc depended on CD8+ T cells as well as NK cells. Furthermore, the combination of GITRL-Fc with PD-1 blockade significantly reduced the tumor growth in the Renca murine kidney adenocarcinoma tumor model. Taken together, these results suggest that GITRL-Fc can improve cancer treatment as a single agent or in combination therapy by enhancing innate and adaptive cellular immunity. Citation Format: Hyun-Bae Jie, Minu K. Srivastava, Erin Mayes, Rui Yun, Fumiko Axelrod, Jorge Monteon, Austin Gurney, Angie In-Kyung Park. GITRL-Fc can significantly reduce tumor growth by stimulating innate and adaptive immunity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3215.

Abstract 2214: GITR ligand fusion protein (GITRL-Fc) induces T cell mediated anti-tumor immune response and can combine with anti-PDL1 to enhance anti-tumor immunity and long-term immune memory

Abstract GITRL (Glucocorticoid-Induced Tumor Necrosis Factor Receptor Ligand, TNFSF18) is a member of the TNF superfamily and naturally exists as a membrane-anchored type II protein that self assembles as a trimer. GITRL activates the co-stimulatory receptor GITR. GITR is found primarily on activated T effector (Teff) cells and regulatory T (Treg) cells. Co-stimulation of GITR by agonist agents is hypothesized to promote anti-tumor immunity by enhancing Teff cell activity and inhibiting Treg suppression. We generated a novel single-gene GITRL trimer fused to an immunoglobulin Fc domain (GITRL-Fc). GITRL-Fc activated GITR signaling more effectively than prototype GITR agonist antibody DTA-1. GITRL-Fc promoted a robust anti-tumor immune response in multiple syngeneic mouse tumor models. GITRL-Fc enhanced tumor specific T-cell responses, particularly of the Th1 type, and also led to reduction in Treg-mediated immunesuppressive activity. GITRL-Fc displayed single agent activity in inhibiting tumor growth and promoting complete tumor rejection in the murine CT26 colon carcinoma model and combination activity with anti-PDL1 as compared to anti-PDL1 and control IgG2a alone. Mice “cured” with GITRL or GITRL/anti-PDL1 combination treatments were protected from re-challenge with tumor cells, suggesting the existence of immunologic memory. More mice were protected from tumor re-challenge with the combination of GITRL-Fc and anti-PDL1, as compared to GITRL-Fc alone. Our results demonstrate that agonist GITRL-Fc induces potent T cell responses, overcomes Treg inhibition, and promotes anti-tumor activity in preclinical models as a single agent or in combination with anti PDL1. The mechanism of tumor eradication and induction of long-term immune memory response by the combination is under investigation and will be discussed at the presentation. Citation Format: Minu K. Srivastava, Rui Yun, Erin Mayes, Hyun_Bae Jie, Fumiko Axelrod, Jorge Monteon, Ming-Hong Xie, John Lewicki, Tim Hoey, Austin Gurney, Angie Inkyung Park. GITR ligand fusion protein (GITRL-Fc) induces T cell mediated anti-tumor immune response and can combine with anti-PDL1 to enhance anti-tumor immunity and long-term immune memory. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2214.

First-in-human phase 1 single-dose study of TRX-518, an anti-human glucocorticoid-induced tumor necrosis factor receptor (GITR) monoclonal antibody in adults with advanced solid tumors.

3017Background: TRX518 is a novel, fully humanized agylcosyl IgG1 anti-GITR mAb. GITR is a member of the tumor necrosis factor receptor family expressed on T, B, & NK cells, and antigen presenting cells. GITR is expressed at high levels by Tregs and up-regulated following T cell activation; minimally expressed by nazve CD4+ and CD8+ T cells. Signaling through GITR abrogates Treg mediated suppression and enhances CD4+ and CD8+ T cell proliferation and TCR stimulation. GITR signaling may enhance host immune responses against tumor and aid in tumor rejection. This study evaluated the safety of a single dose of TRX518 in pts with advanced refractory solid tumors. Methods: Pts received a single dose of TRX518 on Day 1; doses ranged from 0.0001 to 8 mg/kg in 9 cohorts of up to 6 pts/cohort. The study was to determine safety and tolerability, maximum tolerated dose (MTD), pharmacokinetic (PK), and pharmacodynamic (PD) profiles of TRX518. Efficacy was evaluated with the immune related response criteria (irRC). Re...

GITRL is associated with increased autoantibody production in patients with rheumatoid arthritis.

The study aimed to determine the serum level of glucocorticoid-induced tumor necrosis factor receptor family-related protein ligand (GITRL) in patients with rheumatoid arthritis (RA) and investigate its clinical significance. GITRL levels were measured by enzyme-linked immunosorbent assay (ELISA) in 88 RA patients, 20 osteoarthritis (OA) patients, and 20 healthy controls (HCs). Anti-cyclic citrullinated peptide (anti-CCP) antibodies and rheumatoid factor immunoglobulin G (RF-IgG) were also tested by ELISA. RF-IgM, anti-keratin antibody (AKA), and anti-perinuclear factor (APF) antibodies and the erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), and immunoglobulins were measured by standard laboratory techniques. The disease activity was evaluated by the 28-joint count Disease Activity Score (DAS28). GITRL concentrations were significantly elevated in both serum and synovial fluid (SF) of RA patients. GITRL levels in RA sera were significantly higher than those in matched SFs. Positive correlations were found between serum GITRL levels and inflammation parameters or autoantibody production. GITRL levels are significantly elevated in RA serum and SF and are positively correlated with autoantibody production in RA, suggesting a role of GITRL in the development of RA.

GITR Activation Positively Regulates Immune Responses against Toxoplasma gondii.

Toxoplasma gondii is a widespread parasite responsible for causing clinical diseases especially in pregnant and immunosuppressed individuals. Glucocorticoid-induced TNF receptor (GITR), which is also known as TNFRS18 and belongs to the TNF receptor superfamily, is found to be expressed in various cell types of the immune system and provides an important costimulatory signal for T cells and myeloid cells. However, the precise role of this receptor in the context of T. gondii infection remains elusive. Therefore, the current study investigated the role of GITR activation in the immunoregulation mechanisms induced during the experimental infection of mice with T. gondii. Our data show that T. gondii infection slightly upregulates GITR expression in Treg cells and B cells, but the most robust increment in expression was observed in macrophages and dendritic cells. Interestingly, mice infected and treated with an agonistic antibody anti-GITR (DTA-1) presented a robust increase in pro-inflammatory cytokine production at preferential sites of parasite replication, which was associated with the decrease in latent brain parasitism of mice under treatment with DTA-1. Several in vivo and in vitro analysis were performed to identify the cellular mechanisms involved in GITR activation upon infection, however no clear alterations were detected in the phenotype/function of macrophages, Tregs and B cells under treatment with DTA-1. Therefore, GITR appears as a potential target for intervention during infection by the parasite Toxoplasma gondii, even though further studies are still necessary to better characterize the immune response triggered by GITR activation during T. gondii infection.

Ptpn22 Modifies Regulatory T Cell Homeostasis via GITR Upregulation.

PTPN22 gene variation associates with multiple autoimmune diseases, including type 1 diabetes and rheumatoid arthritis. Loss of function studies have demonstrated that PTPN22 impinges on the homeostatic behavior of regulatory T (Treg) cells, a lineage critical for immune tolerance. The frequency and absolute number of Treg cells is increased in Ptpn22-deficient mice, but the mechanism driving this increase is unknown. In this study, we show that Ptpn22 knockdown (KD) promoted the expansion of the Treg cell compartment by upregulating the glucocorticoid-induced TNFR family-related protein (GITR) and increasing GITR signaling. Ptpn22 KD did not accelerate cell division but instead prolonged Treg cell survival, as measured by a decrease in the frequency of apoptotic Treg cells. Loss of Ptpn22 caused a concomitant increase in the proportion of CD44(hi)CD62L(lo) effector Treg cells, at the expense of CD44(lo)CD62L(hi) central Treg cells. The increase in Treg cell numbers, but not their differentiation toward an effector phenotype, was dependent on GITR signaling, because blockade of GITR ligand prevented Treg cell expansion caused by Ptpn22 KD. These findings indicate that GITR plays a key role in regulating the overall size of the Treg cell pool. Our results suggest that the size and composition of the Treg cell compartment are independently controlled and have implications for the design of immunotherapies that seek to improve Treg cell function.

Decidual vascular endothelial cells promote maternal–fetal immune tolerance by inducing regulatory T cells through canonical Notch1 signaling

Adaptation of the maternal immune response to accommodate the semiallogeneic fetus is necessary for pregnancy success. However, the mechanisms by which the fetus avoids rejection despite expression of paternal alloantigens remain incompletely understood. Regulatory T cells (Treg cells) are pivotal for maintaining immune homeostasis, preventing autoimmune disease and fetus rejection. In this study, we found that maternal decidual vascular endothelial cells (DVECs) sustained Foxp3 expression in resting Treg cells in vitro. Moreover, under in vitro Treg cell induction condition with agonistic antibodies and transforming growth factor (TGF)-β, DVECs promoted Treg cell differentiation from non-Treg conventional T cells. Consistent with the promotion of Treg cell maintenance and differentiation, Treg cell-associated gene expression such as TGF-β, Epstein-Barr-induced gene-3, CD39 and glucocorticoid-induced tumor necrosis factor receptor was also increased in the presence of DVECs. Further study revealed that DVECs expressed Notch ligands such as Jagged-1, Delta-like protein 1 (DLL-1) and DLL-4, while Treg cells expressed Notch1 on their surface. The effects of DVECs on Treg cells was inhibited by siRNA-induced knockdown of expression of Jagged-1 and DLL-1 in DVECs. Downregulation of Notch1 in Treg cells using lentiviral shRNA transduction decreased Foxp3 expression in Treg cells. Adoptive transfer of Notch1-deficient Treg cells increased abortion rate in a murine semiallogeneic pregnancy model. Taken together, our study suggests that maternal DVECs are able to maintain decidual Treg cell identity and promote Treg cell differentiation through activation of Notch1 signal pathway in Treg cells and subsequently inhibit the immune response against semiallogeneic fetuses and preventing spontaneous abortion.

Abstract B183: Treg inhibition by anti-GITR therapy evokes therapeutic antitumor responses selectively in the context of a pro-inflammatory tumor microenvironment

Abstract The recent clinical successes of CTLA4 and PD-1 blockade have clearly demonstrated the therapeutic efficacy to enhance anticancer immune responses. Novel targets of immunomodulation are now under investigation to increase the number of immunotherapeutic options available for relapsing/refractory patients. Activation of co-stimulatory molecules with agonist monoclonal antibody (mAbs) represents the most rationale alternative option. Based on the impressive preclinical activity of stimulating the glucocorticoid-induced TNF receptor (GITR) with the mAb DTA-1, an agonist anti-human GITR mAb has very recently entered the clinical evaluation. Although DTA-1, as a monotherapy, proved effective in curing established immunogenic tumors, it failed against advanced poorly immunogenic models, such as B16 melanoma. As anti-GITR therapy is translated into clinic, defining the basis of its activity and failure is key to identify prognostic markers and more effective combination therapies. We thus studied how GITR stimulation with the agonist antibody DTA-1 affects T cells in B16 melanoma-bearing mice treated in curative (early) or refractory (advanced) condition, and verified their functional changes in vitro. In vivo, the comparison of advanced versus early B16 microenvironment before treatment revealed a significant increase in Treg frequency, which caused the Teff/Treg ratio to decrease, and a dysfunctional Teff phenotype. Moreover, B16 cells, in the setting of established tumors, expressed lower levels of MHC-I and MHC-II molecules. DTA-1 consistently reduced intratumor Treg frequency and expression of suppression markers in both curative and refractory conditions. However, Teff activation and tumor-associated MHCs were induced only when DTA-1 was administered in curative conditions. In vitro, pre-incubation of Tregs with DTA-1 completely abolished their ability to inhibit proliferation of target Teff, without affecting Treg expression of the lineage markers Foxp3 and Helios. In addition, treatment with DTA-1, in comparison with the matched IgG isotype control, boosted the release of IFN-gamma, TNF-alpha, IL-17 and IL-6 cytokines in cultures of Tregs, pointing to the promotion of a Th1/Th17 differentiation profile following GITR engagement. Interestingly, when B16 cells were added in culture with Tregs and incubated with DTA-1, they up-regulated MHC-I and MHC-II molecules in a Treg and/or DTA-1 dependent manner. The presence of tumor cells during exposure of Tregs to anti-GITR stimulation did not further alter their function and phenotype, indicating a major direct effect of DTA-1 on Tregs. To confirm the functional modulation of GITR stimulation in human Tregs, we tested their activity in vitro upon pre-incubation with a recombinant human GITR ligand (GITRL). We consistently found that proliferation of activated Teff was no longer suppressed if Tregs were previously treated with GITRL. These results validate GITR as a target to revert immune tolerance in the human setting. Our data indicate that anti-GITR therapy counteracts Treg-mediated immunosuppression independently from the stage of the disease, whereas T-cell activation and tumor immune recognition are favored selectively in the presence of low tumor burden. These findings suggest that, in the setting of advanced and poorly immunogenic tumors, anti-GITR therapy should be exploited in combination with treatments promoting Teff functions and/or tumor killing. Citation Format: Roberta Zappasodi, Sadna Budhu, Cailian Liu, Kyle Draleau, David Schaer, Hong Zhong, Xia Yang, Taha Merghoub, Jedd D. Wolchok. Treg inhibition by anti-GITR therapy evokes therapeutic antitumor responses selectively in the context of a pro-inflammatory tumor microenvironment. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B183.