A Novel Murine GITR Ligand Fusion Protein Induces Antitumor Activity as a Monotherapy That Is Further Enhanced in Combination with an OX40 Agonist.

Purpose: To generate and characterize a murine GITR ligand fusion protein (mGITRL-FP) designed to maximize valency and the potential to agonize the GITR receptor for cancer immunotherapy.Experimental Design: The EC50 value of the mGITRL-FP was compared with an anti-GITR antibody in an in vitro agonistic cell-based reporter assay. We assessed the impact of dose, schedule, and Fc isotype on antitumor activity and T-cell modulation in the CT26 tumor model. The activity of the mGITRL-FP was compared with an agonistic murine OX40L-FP targeting OX40, in CT26 and B16F10-Luc2 tumor models. Combination of the mGITRL-FP with antibodies targeting PD-L1, PD-1, or CTLA-4 was analyzed in mice bearing CT26 tumors.Results: The mGITRL-FP had an almost 50-fold higher EC50 value compared with an anti-murine GITR antibody. Treatment of CT26 tumor-bearing mice with mGITRL-FP-mediated significant antitumor activity that was dependent on isotype, dose, and duration of exposure. The antitumor activity could be correlated with the increased proliferation of peripheral CD8+ and CD4+ T cells and a significant decrease in the frequency of intratumoral Tregs. The combination of mGITRL-FP with mOX40L-FP or checkpoint inhibitor antagonists enhanced antitumor immunity above that of monotherapy treatment.Conclusions: These results suggest that therapeutically targeting GITR represents a unique approach to cancer immunotherapy and suggests that a multimeric fusion protein may provide increased agonistic potential versus an antibody. In addition, these data provide, for the first time, early proof of concept for the potential combination of GITR targeting agents with OX40 agonists and PD-L1 antagonists. Clin Cancer Res; 23(13); 3416-27. ©2017 AACR.

Anti-GITR agonist therapy intrinsically enhances CD8 T cell responses to chronic lymphocytic choriomeningitis virus (LCMV), thereby circumventing LCMV-induced downregulation of costimulatory GITR ligand on APC.

The costimulatory TNFR family member GITR can provide important survival signals for CD8 T cells. However, little is known about the regulation of this pathway during a chronic infection. In this study, we show that GITR ligand (GITRL) is maximally induced on APCs at day 2 post-lymphocytic choriomeningitis virus (LCMV) clone 13 infection, but is downregulated to below baseline levels by day 8 postinfection (p.i.), and remains so at the chronic stage of infection. At its peak, GITRL expression is highest on macrophages, with lower expression on conventional and plasmacytoid dendritic cells. GITR expression was highest on T regulatory cells but was also detected on Th1 and LCMV-specific CD8 T cells at day 8 p.i. and was maintained at low, but above baseline levels at the chronic stage of LCMV infection. As GITRL was limiting at the chronic stage of infection, we investigated the potential of therapeutic stimulation of GITR at this stage using agonistic anti-GITR Ab. Anti-GITR treatment at day 21 p.i. increased the frequency and number of LCMV-specific CD8 T cells, resulting in increased in vivo CTL activity and a concomitant decrease in viral load, despite the persistence of PD-1 expression. These effects of anti-GITR were CD8 T cell intrinsic, with no detectable effects on Th1 or T regulatory cells. In contrast to other TNFR agonists, such as anti-4-1BB, which can cause immune pathology, a single therapeutic dose of anti-GITR did not induce splenomegaly or increase serum alanine transaminase. These studies identify GITR as a promising therapeutic target for chronic infection.

Metformin increases the novel adipokine cartonectin/CTRP3 in women with polycystic ovary syndrome.

CONTEXT: Recently cartonectin was reported as a novel adipokine, with lower levels in diet-induced obese mice, glucose-lowering effects, and antiinflammatory and cardioprotective properties. Polycystic ovary syndrome (PCOS) is a proinflammatory state associated with obesity, diabetes, dyslipidemia, and cardiovascular complications. OBJECTIVES: The objective of the study was to investigate cartonectin levels and regulation in sera and adipose tissue (AT) as well as the effects of metformin of women with PCOS and control subjects. DESIGN: This was a cross-sectional study [PCOS (n = 83) and control (n = 39) subjects]. Real-time PCR and Western blotting were used to assess mRNA and protein expression of cartonectin. Serum cartonectin was measured by an ELISA. RESULTS: Serum and omental adipose tissue cartonectin were significantly lower in women with PCOS compared with control subjects (P < .05 and P < .01, respectively). Furthermore, cartonectin showed a significant negative association with body mass index, waist to hip ratio, glucose, insulin, total cholesterol, low-density lipoprotein-cholesterol, triglycerides, High sensitivity C-reactive protein (hs-CRP) and intima-media thickness (P < .05 and P < .01, respectively); in multiple regression analyses, triglycerides (P =.040) and hs-CRP (P = .031) were predictive of cartonectin levels (P < .05). After 6 months of metformin treatment, there was an associated increase in serum cartonectin (P < .05). Importantly, changes in hs-CRP were significantly negatively correlated with changes in serum cartonectin (P = .033). Finally, cartonectin protein production and secretion into conditioned media were significantly increased by metformin in control human omental AT explants (P < .05). CONCLUSIONS: Serum and omental AT cartonectin are lower in women with PCOS. Metformin treatment increases serum cartonectin levels in these women and in omental AT explants.

Glucocorticoid-induced tumor necrosis factor receptor family-related ligand triggering upregulates vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 and promotes leukocyte adhesion.

The interaction of glucocorticoid-induced tumor necrosis factor receptor-family related (GITR) protein with its ligand (GITRL) modulates different functions, including immune/inflammatory response. These effects are consequent to intracellular signals activated by both GITR and GITRL. Previous results have suggested that lack of GITR expression in GITR(-/-) mice decreases the number of leukocytes within inflamed tissues. We performed experiments to analyze whether the GITRL/GITR system modulates leukocyte adhesion and extravasation. For that purpose, we first evaluated the capability of murine splenocytes to adhere to endothelial cells (EC). Our results indicated that adhesion of GITR(-/-) splenocytes to EC was reduced as compared with wild-type cells, suggesting that GITR plays a role in adhesion and that this effect may be due to GITRL-GITR interaction. Moreover, adhesion was increased when EC were pretreated with an agonist GITR-Fc fusion protein, thus indicating that triggering of GITRL plays a role in adhesion by EC regulation. In a human in vitro model, the adhesion to human EC of HL-60 cells differentiated toward the monocytic lineage was increased by EC pretreatment with agonist GITR-Fc. Conversely, antagonistic anti-GITR and anti-GITRL Ab decreased adhesion, thus further indicating that GITRL triggering increases the EC capability to support leukocyte adhesion. EC treatment with GITR-Fc favored extravasation, as demonstrated by a transmigration assay. Notably, GITRL triggering increased intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression and anti-ICAM-1 and anti-VCAM-1 Abs reversed GITR-Fc effects. Our study demonstrates that GITRL triggering in EC increases leukocyte adhesion and transmigration, suggesting new anti-inflammatory therapeutic approaches based on inhibition of GITRL-GITR interaction.

Therapeutic targeting of the TNF superfamily: a promising treatment for advanced endometrial adenocarcinoma.

Surgical treatment including total abdominal hysterectomy+bilateral salpingo oopherectomy (TAH+BSO) with pelvic and para-aortic lymphadenectomy may not be sufficient to treat cases with advanced endometrial adenocarcinoma (EAC), and in these cases, adjuvant treatments including radiotherapy and/or chemotherapy, are employed based upon the tumor location, type and stage of the disease. These treatment modalities have high incidence of systemic toxicity, thereby compelling clinicians to look for targeted therapy aiming specifically at the malignant cells. Bevacizumab (anti-VEGF), temsirolimus (mTOR inhibitor) and aflibercept (VEGF trap) are already under clinical trials in women with EAC. Targeting the ligands and receptors of the tumor necrosis factor (TNF) superfamily holds promise in this regard. The objective of this review is to provide an overview of the various mechanisms and pathways related to the TNF superfamily involved in advanced EAC and to identify the new therapeutic strategies for specifically targeting these impaired pathways. In addition, the development of treatments for EAC is also discussed. The possible therapeutic treatments include targeting TNFα and its receptors using monoclonal antibodies (MAbs) such as infliximab, adalimumab, etanercept, and certolizumab. Proteosome inhibitors including bortezomib and the anti CD-20 agent rituximab are used to inhibit the NF-κB pathway. Other options include targeting the FAS (CD95) pathway and the TNF-related apoptosis-inducing ligand (TRAIL) pathway using agents such as mapatumab, lexatumumab, and conatumumab. These pathways are known to be involved in the pathogenesis of EAC. Moreover, there is adequate evidence to warrant the use of drugs that target the TNF superfamily for the treatment of advanced EAC.

Pharmacological modulation of GITRL/GITR system: therapeutic perspectives.

Glucocorticoid-induced TNFR-related (gitr) is a gene coding for a member of the TNF receptor superfamily. GITR activation by its ligand (GITRL) influences the activity of effector and regulatory T cells, thus participating in the development of immune response against tumours and infectious agents, as well as in autoimmune and inflammatory diseases. Notably, treating animals with GITR-Fc fusion protein ameliorates autoimmune/inflammatory diseases while GITR triggering, by treatment with anti-GITR mAb, is effective in treating viral, bacterial and parasitic infections, as well in boosting immune response against tumours. GITR modulation has been indicated as one of the top 25 most promising research areas by the American National Cancer Institute, and a clinical trial testing the efficacy of an anti-GITR mAb in melanoma patients has been started. In this review, we summarize results regarding: (i) the mechanisms by which GITRL/GITR system modulates immune response; (ii) the structural and functional studies clearly demonstrating differences between GITRL/GITR systems of mice and humans; (iii) the molecules with pharmacological activities including anti-GITR mAbs, GITR-Fc and GITRL-Fc fusion proteins, GITRL in monomer or multimer conformation; and (iv) the possible risks deriving from GITRL/GITR system pharmacological modulation. In conclusion, GITR triggering and inhibition could be useful in treating tumours, infectious diseases, as well as autoimmune and inflammatory diseases. However, differences between mouse and human GITRL/GITR systems suggest that further preclinical studies are needed to better understand how safe therapeutic results can be obtained and to design appropriate clinical trials.

Technical advance: soluble OX40 molecule mimics regulatory T cell modulatory activity on FcεRI-dependent mast cell degranulation.

Tregs play a central role in modulating FcεRI-dependent MC effector functions in the course of the allergic response. Cellular interaction depends on the constitutive expression of OX40 on Tregs and the OX40L counterpart on MCs. Study of OX40L signaling on MCs is hampered by the need of a highly purified molecule, which triggers OX40L specifically. We now report that sOX40 mimics the physiological activity of Treg interaction by binding to activated MCs. When treated with sOX40, activated MCs showed decreased degranulation and Ca(++) influx, whereas PLC-γ2 phosphorylation remained unaffected. Once injected into experimental animals, sOX40 not only located within the endothelium but also in parenchyma, where it could be found in close proximity and apparently bound to MCs. This soluble molecule triggers MC-OX40L without the requirement of Tregs, thus allowing study of OX40L signaling pathways in MCs and in other OX40L-expressing cell populations. Importantly, as sOX40 inhibits MC degranulation, it may provide an in vivo therapeutic tool in allergic disease.

The role of TNF superfamily members in T-cell function and diseases.

Interactions that occur between several tumour necrosis factor (TNF)-TNF receptors that are expressed by T cells and various other immune and non-immune cell types are central to T-cell function. In this Review, I discuss the biology of four different ligand-receptor interactions - OX40 ligand and OX40, 4-1BB ligand and 4-1BB, CD70 and CD27, and TL1A and death receptor 3 - and their potential to be exploited for therapeutic benefit. Manipulating these interactions can be effective for treating diseases in which T cells have an important role, including inflammatory conditions, autoimmunity and cancer. Here, I explore how blocking or inducing the signalling pathways that are triggered by these different interactions can be an effective way to modulate immune responses.

OX40 agonist therapy enhances CD8 infiltration and decreases immune suppression in the tumor.

Acquisition of full T-cell effector function and memory differentiation requires appropriate costimulatory signals, including ligation of the costimulatory molecule OX40 (TNFRSF4, CD134). Tumors often grow despite the presence of tumor-specific T cells and establish an environment with weak costimulation and immune suppression. Administration of OX40 agonists has been shown to significantly increase the survival of tumor-bearing mice and was dependent on the presence of both CD4 and CD8 T cells during tumor-specific priming. To understand how OX40 agonists work in mice with established tumors, we developed a model to study changes in immune cell populations within the tumor environment. We show here that systemic administration of OX40 agonist antibodies increased the proportion of CD8 T cells at the tumor site in three different tumor models. The function of the CD8 T cells at the tumor site was also increased by administration of OX40 agonist antibody, and we observed an increase in the proportion of antigen-specific CD8 T cells within the tumor. Despite decreases in the proportion of T regulatory cells at the tumor site, T regulatory cell function in the spleen was unaffected by OX40 agonist antibody therapy. Interestingly, administration of OX40 agonist antibody caused significant changes in the tumor stroma, including decreased macrophages, myeloid-derived suppressor cells, and decreased expression of transforming growth factor-beta. Thus, therapies targeting OX40 dramatically changed the tumor environment by enhancing the infiltration and function of CD8 T cells combined with diminished suppressive influences within the tumor.

GITR ligand-costimulation activates effector and regulatory functions of CD4+ T cells.

Engagement of glucocorticoid-induced TNFR-related protein (GITR) enables the costimulation of both CD25(-)CD4(+) effector (Teff) and CD25(+)CD4(+) regulatory (Treg) cells; however, the effects of GITR-costimulation on Treg function remain controversial. In this study, we examined the effects of GITR ligand (GITRL) binding on the respective functions of CD4(+) T cells. GITRL-P815 transfectants efficiently augmented anti-CD3-induced proliferation and cytokine production by Teff cells. Proliferation and IL-10 production in Treg were also enhanced by GITRL transfectants when exogenous IL-2 and stronger CD3 stimulation was provided. Concomitant GITRL-costimulation of Teff and Treg converted the anergic state of Treg into a proliferating state, maintaining and augmenting their function. Thus, GITRL-costimulation augments both effector and regulatory functions of CD4(+) T cells. Our results suggest that highly activated and increased ratios of Treg reverse the immune-enhancing effects of GITRL-costimulation in Teff, which may be problematic for therapeutic applications using strong GITR agonists.

Administration of agonistic anti-4-1BB monoclonal antibody leads to the amelioration of inflammatory bowel disease.

4-1BB (CDw 137), a member of the tumor necrosis factor receptor (TNFR) superfamily, is a costimulatory receptor primarily expressed on activated T cells. It has been shown that the administration of agonistic anti-4-1BB monoclonal antibody (mAb) enhances tumor immunity and allogenic immune responses. Paradoxically, we found that the administration of anti-4-1BB mAb reduced the incidence and severity of inflammatory bowel disease. In this study, we investigated the effects of anti-4-1BB mAb in a murine intestinal inflammation model, which induced by the hapten reagent, 2,4,6-trinitrobenzene sulfonic acid (TNBS) and mimics immunologic characteristics of human Crohn's disease (CD). Colitis was induced by rectal administration of 2mg of TNBS in 35% ethanol using a vinyl catheter positioned 4cm from the anus. All mice were sacrificed 3 and 10 days after the TNBS administration. The disease activity index (DAI), histological changes of the colon and production of cytokines (IL-2, IL-4, IL-10 and IFN-gamma) were evaluated. The surface molecules of T cells in peripheral blood, spleen and mesenteric lymph nodes were analyzed by flow cytometry. When mice were treated with anti-4-1BB mAb, improvement in both wasting and histopathologic signs of colonic inflammation was observed. The increase a number of splenic CD4(+)CD25(+) T cells and decreased synthesis of the Th1 cytokine IL-2 also occurred. Interestingly, increased production of Th1 cytokine IFN-gamma and proportion of CD8(+) T cells were observed in mice treated with anti-4-1BB mAb in comparison to the colitic mice. These studies show, for the first time, that agonistic anti-4-1BB mAb can improve experimental colitis by reduction of IL-2 and augmentation of CD4(+)CD25(+) regulatory T cells. TNBS colitis is Th1-mediated and has similar histologic features and distribution of inflammation to CD. This study suggests that anti-4-1BB mAb therapy could be effective in the treatment of patients with CD.