GITR Ligand Research Articles

Identification of T regulatory cell (Treg) proliferation induced by IgM+IgDlo regulatory B cell (BDL) using a multi-color immunofluorescence strategy

Abstract Multiple sclerosis is an autoimmune disease driven by T cell orchestrated demyelination. CD4+Foxp3+ T regulatory cells (Treg) suppress autoimmunity; thus, therapies to increase endogenous Treg numbers are ideal. We identified a novel subset of regulatory IgM+IgDlow/- B cells (BDL) that induce Treg proliferation in a glucocorticoid-induced TNFR-related ligand (GITRL)-dependent manner via cell-cell contact. However, where BDL localize with Treg in the spleen is unknown and imaging is challenging due to numerous required markers. Because Treg are located in the T cell zone, we hypothesize BDL migrate to and interact with Treg at the T-B boundary. A multi-color panel was developed to visualize, localize, and quantitate BDL-Treg conjugates. Fluorescent antibodies were selected via compensation methods using single-stained controls. Serial splenic sections were used to localize BDL-Treg interactions by staining a section with antibodies for splenic architecture: MAdCAM-1 (marginal zone), IgM (B follicle), and CD4 (T cell zone). The consecutive section was stained with antibodies to identify BDL (IgM, IgD), Treg (CD4, Foxp3), and proliferation (Ki-67). This strategy successfully identified Ki-67+ Treg-BDL conjugates in the T-B boundary. Future studies will quantitate the number of Ki-67+ Treg surrounded by BDL. We hope this strategy can be used to aid the development of a BDL-based therapeutic to increase endogenous Treg numbers and reduce autoimmunity.

T CELLS ENGINEERED TO EXPRESS A CAR ANTI-GD2 AND THE COSTIMULATORY MOLECULE GITRL ERADICATE GLIOBLASTOMA CELLS AND ARE IFNY PRODUCERS

The adoptive transfer of T lymphocytes expressing chimeric antigen receptors (CAR) has resulted in impressive complete remission rates in B-cell malignancies. However, the therapeutic efficacy of CAR-T cells is still low or non-existent against solid tumors, which make up the vast majority of neoplasms. One of the main reasons for this failure is the presence of ligands and immunosuppressive cells in the tumor microenvironment. The complete activation of T cells requires the engagement of costimulatory molecules whose expression is temporally segregated and whose nature of biochemical signals are complementary. This is the case of CD28, expressed constitutively, and GITR, expressed right after the initial activation of T cells. Therefore, our hypothesis is that the combined expression of a CAR containing the costimulatory domain CD28 and the GITR ligand (GITRL) will potentiate the antineoplastic action of CAR-T cells. In addition to providing complementary costimulatory signals for effector CAR-T cells, GITRL can suppress the action of regulatory T cells in the tumor microenvironment, thus possibly reducing local immunosuppression. Then, the aim of this study is to evaluate the therapeutic efficiency of anti-GD2 CAR-T cells coexpressing GITRL in a preclinical model of glioblastoma multiforme. In this project, we used as a model a CAR against the ganglioside GD2, which is highly immunogenic and expressed in tumors of neuroectodermal origin, such as glioblastoma. To test our hypothesis, we generated two lentiviral vectors to express CAR anti-GD2 and another one coexpressing GITRL as a costimulatory domain. After that, we produced lentiviral particles to transduced human T lymphocytes and we detected CAR expression in the T cells transduced with both vectors. To verify the functional activity of these CAR-T cells, we performed a coculture assay against T98G cell line, which express high levels of GD2, and HCT-166 cell, that dont express. These cell lines were previously modified to express luciferase to enable the analysis through bioluminescence imaging. We tested two ratios of effector:target cells (E:T) and we observed that both CAR.GD2 and CAR.GD2-GITRL T cells demonstrated powerful capacity of lysis only against cells that express GD2. Then, we interrogated whether these CAR-T cells could keep their lysis capacity after additional rechallenges. So, another two rounds of coculture were performed and the cells showed persistence of cytotoxicity. In addition, tumor cell killing was associated with secretion of IFNy as evaluated by ELISA quantification of coculture supernatants. These data encouraged us to compare the antineoplastic activity and persistence of CAR-T cells anti-GD2 in an orthotopic pre-clinical model of glioblastoma multiforme, which is under way. We expect that the potential results of this project will foster the development of a new advanced cellular immunotherapy strategy for the treatment of solid GD2+ neoplasms and will pave the way to increase the antineoplastic efficiency of CAR-T cells against other malignancies.

Neoadjuvant Chemoradiotherapy Changes the Landscape of Soluble Immune Checkpoint Molecules in Patients With Locally Advanced Rectal Cancer.

BackgroundWe aimed to investigate clinical implications of specific soluble immune checkpoint molecules (sICMs) in locally advanced rectal cancer (LARC) patients treated with neoadjuvant chemoradiotherapy (nCRT).MethodsWe prospectively enrolled 30 LARC patients treated with nCRT and collected blood samples from them before, during, and after nCRT for prospective studies. Immune checkpoints often refer to T cell surface molecules influencing the immune response. Immune checkpoints, in the form of a soluble monomeric form, is widely present in blood. In the study, eight immune checkpoint-related plasma proteins, including programmed death-ligand 1 (PD-L1), CD80, CD86, CD28, CD27, glucocorticoid-induced tumor necrosis factor receptor (GITR), GITR ligand (GITRL), and inducible T-cell costimulator (ICOS), were measured using the Luminex platform. Two independent pathologists categorized patients as the good responders and the poor responders according to Dworak tumor regression grade (TRG).ResultsOf the 30 patients, the levels of sPD-L1, sCD80, sCD86, sCD28, sGITR, sGITRL, sCD27, and sICOS decreased during nCRT (Pre-nCRT vs. During-nCRT, all p<0.05) but were restored after nCRT treatment (Pre-nCRT vs. Post-nCRT, all p>0.05). In the 14 good responders, the levels of sICMs, other than sGITR (p=0.081) and sGITRL (p=0.071), decreased significantly during nCRT (Pre-nCRT vs. During-nCRT, p<0.05), but they were all significantly increased after nCRT (During-nCRT vs. Post-nCRT, all p<0.05). In the 16 poor responders, only sCD80 was significantly reduced during nCRT (Pre-nCRT vs. During-nCRT, p<0.05), and none was significantly increased after nCRT (During-nCRT vs. Post-nCRT, all p<0.05). High levels of sICMs before nCRT were associated with poor response (all OR≥1). The Pre-model that incorporated the 8 sICMs before nCRT yielded a good predictive value (AUC, 0.848) and was identified as an independent predictor of treatment response (OR, 2.62; 95% CI, 1.11-6.18; p=0.027).ConclusionOur results suggest chemoradiotherapy could influence the change of sPD-L1, sCD80, sCD86, sCD28, sGITR, sGITRL, sCD27, and sICOS in patients with LARC. The levels of the majority of soluble immune checkpoint molecules were reduced during nCRT and then restored at the end of nCRT, particularly in patients who responded well to nCRT. Combined baseline sICMs can be developed to predict treatment response.

An anti-PD-1\u2013GITR-L bispecific agonist induces GITR clustering-mediated T cell activation for cancer immunotherapy

Costimulatory receptors such as glucocorticoid-induced tumor necrosis factor receptor–related protein (GITR) play key roles in regulating the effector functions of T cells. In human clinical trials, however, GITR agonist antibodies have shown limited therapeutic effect, which may be due to suboptimal receptor clustering-mediated signaling. To overcome this potential limitation, a rational protein engineering approach is needed to optimize GITR agonist-based immunotherapies. Here we show a bispecific molecule consisting of an anti-PD-1 antibody fused with a multimeric GITR ligand (GITR-L) that induces PD-1-dependent and FcγR-independent GITR clustering, resulting in enhanced activation, proliferation and memory differentiation of primed antigen-specific GITR+PD-1+ T cells. The anti-PD-1–GITR-L bispecific is a PD-1-directed GITR-L construct that demonstrated dose-dependent, immunologically driven tumor growth inhibition in syngeneic, genetically engineered and xenograft humanized mouse tumor models, with a dose-dependent correlation between target saturation and Ki67 and TIGIT upregulation on memory T cells. Anti-PD-1–GITR-L thus represents a bispecific approach to directing GITR agonism for cancer immunotherapy.

Therapeutic antibody activation of the glucocorticoid-induced TNF receptor by a clustering mechanism.

GITR is a TNF receptor, and its activation promotes immune responses and drives antitumor activity. The receptor is activated by the GITR ligand (GITRL), which is believed to cluster receptors into a high-order array. Immunotherapeutic agonist antibodies also activate the receptor, but their mechanisms are not well characterized. We solved the structure of full-length mouse GITR bound to Fabs from the antibody DTA-1. The receptor is a dimer, and each subunit binds one Fab in an orientation suggesting that the antibody clusters receptors. Binding experiments with purified proteins show that DTA-1 IgG and GITRL both drive extensive clustering of GITR. Functional data reveal that DTA-1 and the anti-human GITR antibody TRX518 activate GITR in their IgG forms but not as Fabs. Thus, the divalent character of the IgG agonists confers an ability to mimic GITRL and cluster and activate GITR. These findings will inform the clinical development of this class of antibodies for immuno-oncology.

IMMU-13. COMBINED MODULATION OF THE TGF-Β PATHWAY AND GITR IMMUNE CHECKPOINT SIGNALING PROMOTES ANTI-TUMOR IMMUNITY IN SYNGENEIC GLIOMA MODELS

Abstract The profound local immunosuppressive microenvironment is one hallmark of glioblastoma, which results in resistance to most immunotherapeutic strategies that have been explored so far. Reverting this condition in order to reinvigorate anti-glioma immunity might be a promising therapeutic approach. Transforming growth factor (TGF)-β signaling is deregulated in different cancer types and contributes to the malignant phenotype of glioma cells. Glioma-derived TGF-β is also a major immunosuppressive factor in the tumor microenvironment. Furthermore, intratumoral regulatory T (Treg) cells and activated T effector cells express high levels of the co-stimulatory immune checkpoint glucocorticoid-induced tumor necrosis factor receptor (GITR). Agonistic anti-GITR antibodies have been explored in preclinical tumor models and are under investigation in clinical trials for the treatment of solid tumors. We evaluated the effect of TGF-β and GITR targeting on anti-tumor immune responses in syngeneic mouse glioma models. In co-culture settings, GITR modulation with a GITR ligand (GITRL)-Fc fusion protein, given alone or in combination with a pharmacological TGF-β receptor inhibitor, led to increased T cell activation. Furthermore, the combined targeting of the two pathways resulted in significantly higher immune cell-mediated tumor cell killing than either treatment alone. In vivo, TGF-β inhibition and GITR signaling modulation resulted in a higher fraction of long-term surviving glioma-bearing mice than single-agent treatment. Surviving mice were resistant to tumor re-challenge, suggesting adaptive immunity as an underlying mechanism. These data support the assumption that combined immunotherapeutic strategies may represent a promising approach for the treatment of glioma.

Research advances in the role of the glucocorticoid-induced tumor necrosis factor receptor-related protein and its ligand in liver diseases

Glucocorticoid-induced tumor necrosis factor receptor (GITR) is a member of the tumor necrosis factor receptor superfamily, and after it specifically binds to GITR ligand (GITRL), the downstream signals mediated by them can not only serve as a costimulatory molecule to promote the proliferation and cytokine secretion of effector T cells, but also affect the proliferation of regulatory T cells and inhibit the function of effector T cells, thereby regulating the inflammatory response and tumor cell-killing effect of effector T cells. GITRL is mainly expressed in antigen- presenting cells and has an intracellular domain, and the binding of GITRL to GITR can affect the function of antigen-presenting cells via receptor/ligand reverse signaling. In liver diseases, GITRL/GITR signal is not only involved in immune rejection after liver transplantation and gene therapy, but also associated with the formation of tumor immune microenvironment and the immune escape of tumor. Further studies are needed to explore the role of GITRL/GITR in the development and progression of other liver diseases.

An Anti-PD-1-GITR Ligand Bispecific Enhances Antitumor Immunity.

A PD-1-directed, multimeric GITR agonist enhances immune cell activation to inhibit tumor growth.

Concurrent Chemoradiotherapy Increases the Levels of Soluble Immune Checkpoint Proteins in Patients with Locally Advanced Cervical Cancer

Concurrent chemoradiotherapy (CCRT) could influence systematic immunity in cancer patients, but its impact on soluble immune checkpoint molecules remain largely unknown. Here, we investigated the dynamic alterations of 16 soluble immune checkpoint molecules in locally advanced cervical cancer (LACC) patients treated with CCRT. Sixty LACC patients treated with CCRT were prospectively enrolled and patients’ blood was collected before, during, and at the end of CCRT. Sixteen soluble immune checkpoint molecules were tested by Luminex platform. Tumor response was evaluated based on RECIST 1.1 guidelines at one month after CCRT. One-way analysis of variance with Bonferroni's post-test or paired t tests were used to evaluate the change of the levels of soluble immune checkpoint molecules. Forty-five (75%) patients experienced complete response (CR), 12 (20%) patients experienced partial response (PR), and 3 (5%) patients experienced stable disease after CCRT. Levels of T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) increased both during and at the end of CCRT (P = .015 and .003, respectively), while inducible T-cell costimulator (ICOS), B7-2, glucocorticoid-induced tumor necrosis factor receptor ligand (GITRL) increased at the end of CCRT (P = .032, .048, and .050, respectively). The other soluble immune checkpoint molecules including CD40 showed no significant change throughout CCRT (all P >0.05). Interestingly, we found that these changes were more significant in patients with CR than patients with PR/SD. Specifically, for patients with CR, levels of TIM-3 were significantly elevated both during and at the end of CCRT (P = .008 and < .001, respectively), while ICOS, B7-2, GITRL were significantly elevated at the end of CCRT (P = .023, .039, and .042, respectively). Meanwhile, CD40 was significantly elevated at the end of CCRT in patients with CR (P = .040). However, patients with PR/SD, no soluble immune checkpoint molecules were significantly changed (all P > 0.05). Our study reveals that CCRT could induce the elevation of the levels of soluble TIM-3, ICOS, B7-2, and GITRL in the blood of patients with LACC. This phenomenon was more obvious in patients with good response to CCRT.

Low-Dose LPS Induces Tolerogenic Treg Skewing in Asthma

The mechanism(s) underlying endotoxin tolerance in asthma remain elusive. As the endotoxin lipopolysaccharide (LPS) affects the expression of the regulatory T-cell (Treg)-suppressive glucocorticoid-induced tumor necrosis factor receptor ligand (GITRL) on antigen-presenting dendritic cells (DCs), we hypothesized that LPS-induced changes in DC GITRL expression may impact Treg-mediated T-helper (Th) cell suppression and the induction of endotoxin tolerance. Here, we propose a novel mechanism by which low-dose LPS inhalation in neonatal mice induces endotoxin tolerance, thereby offering protection from later asthma development. Three-day old wild-type and Toll-like receptor 4 (TLR4)-deficient neonatal mice were exposed to low-dose LPS (1 μg) intranasally for 10 consecutive days prior to ovalbumin (OVA)-induced asthma to better understand the tolerogenic mechanism(s) of low-dose LPS pre-exposure. In vivo findings were validated using in vitro co-culturing studies of primary CD11c+ DCs and CD4+ T-cells with or without low-dose LPS pre-exposure before OVA stimulation. Low-dose LPS pre-exposure upregulated the Treg response and downregulated pathogenic Th2 and Th17 responses through promoting apoptosis of Th2 and Th17 cells. Low-dose LPS pre-exposure downregulated DC GITRL expression and T-cell GITR expression. Artificial DC GITRL expression abrogated the tolerogenic Treg-skewing effect of low-dose LPS pre-exposure. Low-dose LPS pre-exposure inhibited TRIF/IRF3/IFNβ signaling and upregulated expression of tolerogenic TRIF/IRF3/IFNβ negative regulators in a TLR4-dependent manner. This tolerogenic DC GITRL downregulation was attributable to TRIF/IRF3/IFNβ signaling inhibition. Low-dose LPS pre-exposure produces tolerogenic Treg skewing in neonatal asthmatic mice, a phenomenon attributable to TLR4-dependent TRIF/IRF3/IFNβ-mediated DC GITRL downregulation.

SECTM1A Positively Regulates Tissue-Resident Macrophage Self-Renewal Capacity by Boosting T Helper Cells in Mice During Endotoxemia

Endotoxin-triggered systemic inflammation and multiple organ dysfunction are major culprits to sepsis death. Here, we observed that the plasma levels of SECTM1 in sepsis patients were significantly lower than that in healthy donors. Similarly, the mouse homologous SECTM1A was dramatically down-regulated in multiple organs following LPS injection. Using a global SECTM1A-KO mouse model, we found that SECTM1A depletion exaggerated LPS-caused reduction of tissue-resident macrophage numbers in multi-organs by suppressing their proliferation at earlier stage, leading to more serious organ damage. By contrast, administration of recombinant SECTM1A significantly attenuated macrophage reduction and improved animal survival upon LPS challenge. Mechanistically, we discovered that SECTM1A, as a GITR ligand, contributed to maintaining self-renewal capacity of tissue-resident macrophages during endotoxemia through promoting expansions of T helper cells and releases of macrophage self-renewal cytokines. Our study suggests that SECTM1A is essential for macrophage self-renewal and may serve as a new therapeutic agent for sepsis.

Abstract 4143: The novel hexavalent human GITR agonist HERA-GITRL promotes anti-tumor efficacy independent of Fc-functionality and shows superior activity compared with the monoclonal anti-GITR antibody TRX518

Abstract Glucocorticoid-induced TNFR-related protein (GITR, TNFRSF18, CD357), a TNFR-SF member, is a co-stimulatory receptor that increases anti-tumor T cell activation. Based on Apogenix TNFR-SF agonist HERA-technology, we created a fully human hexavalent GITR ligand fusion protein - HERA-GITRL - intended for T cell costimulatory approaches in immuno-oncology (IO) therapy. HERA-GITRL is composed of a trivalent single chain GITRL-receptor-binding-domain fused to an IgG1-derived silenced Fc-domain serving as dimerization scaffold. The unique design that combines a molecular mimic of the endogenous GITRL with a silenced Fc-domain, allows the study of pure GITR agonism in contrast to Fc-mediated mixed modes of action. Here we report in vitro and in vivo properties of our novel HERA-GITRL construct. For functional characterization of HERA-GITRL in vitro, human immune cells isolated from healthy-donor blood were profiled by multicolor flow cytometry and real-time cell analysis. Stimulation of unfractionated human T cells or purified naïve CD4+ T cells by anti-CD3 antibody was further augmented by HERA-GITRL. This effect was accompanied by increased proliferation, differentiation and elevated levels of TNF-α and IFN-γ. Importantly, HERA-GITRL-mediated T cell activation increases tumor cell killing by PBMCs in vitro and showed in vivo anti-tumor efficacy as a single agent in a subcutaneous syngeneic colon cancer model (CT26wt) in mice. This anti-tumor effect is independent of its Fc functionality, as murine HERA-GITR ligands with functional Fc- or silenced Fc-domains show similar tumor growth inhibition. TRX518, an anti-human GITR monoclonal antibody currently investigated in a clinical Phase I study, was used in a direct in vitro comparison with trivalent GITRL and our hexavalent HERA-GITRL. Without crosslinking HERA-GITRL showed superior agonistic activity over trivalent GITRL and TRX518. Crosslinking increased the activity of trivalent GITRL while the residual activity of TRX518 was even decreased. We constructed a CHO cell line stably expressing fully human HERA-GITRL with high purity and yield. The resulting research cell bank is ready to be used for subsequent GMP process development. By clustering the receptor chains in a spatially well-defined manner, HERA-GITRL induces potent agonistic activity without being dependent on additional Fc-mediated crosslinking. A comparison of HERA-GITRL with the anti-GITR antibody TRX518 showed superior agonistic activity of our HERA construct in vitro with and without cross-linking. The HERA-ligand concept has also been successfully translated to HERA-TRAIL (now in Phase I), -CD40L, -CD27L,-LIGHT and -4-1BBL. Citation Format: Matthias Schröder, Viola Marshall, Meinolf Thiemann, David M. Richards, Christian Merz, Jaromir Sykora, Julian P. Sefrin, Mauricio Redondo-Müller, Karl Heinonen, Katharina Billian-Frey, Oliver Hill, Christian Gieffers. The novel hexavalent human GITR agonist HERA-GITRL promotes anti-tumor efficacy independent of Fc-functionality and shows superior activity compared with the monoclonal anti-GITR antibody TRX518 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4143.

Efficacy of cytokine-induced killer cells targeting CD40 and GITR.

Since the publication of a novel protocol in 1991, cytokine-induced killer (CIK) cells have shown promising results in the treatment against neoplastic diseases. Despite ongoing preclinical and clinical studies, CIK cell treatment in the context of human monoclonal antibodies targeting tumor-necrosis factor receptors remains overlooked. The present study investigated whether a combination of CIK cells with human monoclonal antibody anti-CD40 and anti-Glucocorticoid-induced TNF-related protein (GITR) would lead to further cytotoxicity against tumor cells expressing CD40 and GITR ligand (L). Therefore, in vitro experiments with human lymphoma cell lines SU-DHL-4 and Daudi (both CD40 positive) and human breast adenocarcinoma MCF-7 (GITRL positive) were performed and the secretion of interferon (IFN)-γ was measured. Three interesting results emerged: i) a combination of CIK cells and anti-CD40 mAb is more effective than CIK cell treatment alone; ii) the use of anti-GITR mAb and CIK cells significantly enhanced the cytotoxicity of CIK cells against MCF-7 compared with single CIK cell treatment and iii) the combination of both antibodies and CIK cells abrogates the anti tumoral effect of CIK cells on all three cell lines. By performing an ELISA for IFN-γ measurement, a lower secretion was observed when anti-CD40 or anti-GITR mAb was added. This outcome indicates that further studies in vitro and in vivo may aid in understanding the synergistic molecular mechanisms of CIK cells, and anti-CD40 and anti-GITR mAb.

Identification of Chicken GITR and GITR Ligand, Proof of Their Mutual Interaction, and Analysis of Chicken GITR Tissue Distribution by a Novel Antibody That Reveals Expression on Activated T Cells and Erythrocytes

Glucocorticoid-induced TNFR (GITR) and its ligand, GITRL, belong to the costimulatory members of the TNF superfamily and are crucially involved in the formation and modulation of an effective immune response, comprising innate as well as adaptive mechanisms. In this study, we identify and describe chicken GITR and GITRL, and provide an initial characterization of the newly developed chGITR-specific mAb 9C5. Structural analyses of the putative chicken molecules GITR and GITRL confirmed the conservation of classic topological features compared with their mammalian homologs and suggested the ability of mutual interaction, which was verified via flow cytometry. Whereas only minute populations of native lymphocytes isolated from spleen, bursa, and thymus expressed GITR, it was strongly upregulated upon activation on αβ and γδ T cells, comprising CD4+ as well as CD8+ subsets. In blood, a fraction of CD4+CD25+ T cells constitutively expressed GITR. In addition, virtually all chicken erythrocytes displayed high levels of GITR. Our results verify the existence of both GITR and its ligand, GITRL, in chickens; they provide the basis and novel tools to further characterize their impact within the immune response and reveal the so-far unrecognized expression of GITR on erythrocytes.

Abstract LB-152: Local selivery of GITR agonistic antibody induces a stronger antitumor immunity than systemic administration

Abstract The field of cancer immunotherapy is expanding rapidly with the success of an antagonistic antibody against CTLA-4 and PD-1/PD-L1. However, since approximately half of patients do not respond to the therapies even the combination regimen, the development of novel checkpoint inhibitors is desired for the recurrent or refractory patients. Recently, newer targets including select members of the tumor necrosis factor receptor (TNFR) family, including 4-1BB, OX40 and glucocorticoid-induced tumor necrosis factor receptor (GITR), are gathering attention. GITR is expressed at a low basal level on naïve murine T cells and at a very low level on human T cells, whereas a GITR ligand (GITRL) was abundantly expressed in murine dendritic cells and macrophages. Multiple studies have shown that GITR-GITRL interaction can provide a co-stimulatory signal to both CD4+ and CD8+ naïve T cells, enhancing proliferation and effector function, particularly in the setting of suboptimal T cell receptor stimulation. In contrast, murine and human Tregs constitutively express GITR, and it had been shown that activation of GITR signaling by GITR ligand or agonistic antibody inhibit the suppressive activity of Tregs. To enhance the antitumor effect of immune stimulatory reagents, we have been focusing on the intratumoral administration route. Since the GITR agonistic Ab directly activates effector T cells and suppresses Tregs, the increase of Ab concentration in tumors and surrounding tissues including lymph nodes by the intratumoral route may enhance only the tumor-infiltrating T cells and break the tumor-specific immune-tolerant microenvironment, sparing the unexpected effect on the systemic immune system. In this study, first, in a murine colon cancer model, we showed that the intratumoral delivery of Ab significantly increased the number of effector T cells infiltrated into tumors, and suppressed tumor growth more effectively than the intraperitoneal injection did. Then, we found that the injection of Ab into the peritumoral area induced a systemic antitumor immunity at a similar level to the intratumoral injection. Therefore, we hypothesized that the transfer of locally administrated Ab into tumor-draining lymph nodes (TDLNs) plays an important role in inducing an effective immunity. In fact, intratumorally or peritumorally injected Ab was detected in TDLNs, and resection of Ab-injected TDLNs significantly reduced GITR Ab-mediated systemic tumor immunity. Intratumoral injection showed less number of auto-reactive T cells in the spleen than the intraperitoneal injection did. Furthermore, the AH-1 tetramer assay and ELISpot assay showed that the combination of GITR Ab with anti-PD-1 Ab synergistically enhanced an induction of antitumor immunity, leading to eradication of the tumor. Intratumoral delivery of GITR Ab is a promising approach to induce an effective immunity than the systemic delivery. Citation Format: Kazunori Aoki, Kenta Narumi, Chihiro Shibasaki, Reina Miyakawa. Local selivery of GITR agonistic antibody induces a stronger antitumor immunity than systemic administration [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 LB-152.

Abstract 3826: GITRL-Fc biomarker and mechanism study: GITRL-Fc reduces Treg frequency in tumors and requires effector function for inhibition of tumor growth

Abstract Activation of the co-stimulatory receptor GITR (Glucocorticoid-Induced Tumor Necrosis Factor Receptor) by GITR-Ligand (GITRL) promotes proliferation and activation of effector T cells (T eff) and inhibits suppressive activity of regulatory T cells (Treg). Here, we have further characterized the mechanism of action of a single-gene GITRL trimer fused to an immunoglobulin Fc domain (GITRL-Fc, 336B3) by examining pharmacodynamic (PD) biomarkers in time course studies. Mice bearing CT26.WT colon tumors were treated with weekly GITRL-Fc and sacrificed 24 hours, 7 and 14 days after the first dose. Immuno-phenotyping of tumor-associated immune cells revealed a reduction in Treg frequency in tumor by 24 hours post-dose that was maintained at 7 and 14 days. Furthermore, GITRL-Fc treatment increased activation markers on tumor-associated CD4+ and CD8+ T cells, suggesting an increased cytotoxic environment within the tumor. This was supported by significant and sustained increase in CD8+ T cell:Treg ratio in the tumor after GITRL-Fc treatment. To determine whether intratumoral (IT) injection of GITRL-Fc is an effective route of administration, we compared efficacy, pharmacodynamic (PD) markers and pharmacokinetics in IT- and intraperitoneal (IP)-injected mice bearing bilateral CT26.WT tumors. Both routes of administration showed similar tumor growth inhibition (TGI) and PD markers in both the treated and the abscopal tumors, but IT injection resulted in a significantly lower serum GITRL-Fc concentration, suggesting that IT administration may be an alternative route of administration to IP with similar efficacy. The GITRL-Fc molecule 336B3 is effector function competent and able to induce cell-mediated cytotoxicity upon binding. To determine whether this effector function is required for GITRL-Fc-induced TGI, we treated CT26.WT tumor-bearing mice with 336B3 and 336B22, a GITRL-Fc molecule deficient in effector function. The effector function-competent 336B3 induced significant TGI and a more robust activation of Teff cells and reduction in Treg frequency, when compared to 336B22, suggesting that effector function is important for efficacy. To identify biomarkers for GITRL-Fc, we performed microarray analyses on multiple syngeneic mouse models treated with GITRL-Fc and developed GITRL gene signatures from blood and from tumors. We also developed multiplexed immunohistochemistry panels designed to quantify frequency of GITR and GITRL expression (GITR+CD8, GITR+FOXP3) in tumors. In conclusion, we have examined the effects of GITRL-Fc on preclinical mouse models. Biomarker analysis showed that loss of Tregs, activation of T cells and Fc-mediated effector function are key elements in the mechanism of action of the molecule. We have identified potential biomarkers to be used for PD and potential predictive analysis in clinical trial patient samples. Citation Format: Gretchen M. Argast, Belinda Cancilla, Fiore Cattaruzza, Pete Yeung, Reyhaneh Lahmy, Erwan Le Scolan, Rose Harris, Alayne Brunner, Min Wang, Fumiko Axelrod, Jorge Monteon, Jennifer Elechko, Andrew Lam, MingHong Xie, Earth Light Lowe, Gilbert O'Young, Austin Gurney, Ann M. Kapoun. GITRL-Fc biomarker and mechanism study: GITRL-Fc reduces Treg frequency in tumors and requires effector function for inhibition of tumor growth [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 3826.

Abstract 2726: In vitro functional activity of OMP-336B11, a GITRL-Fc fusion protein, on primary human immune cells

Abstract Glucocorticoid-induced TNFR-related protein (GITR) is a member of the TNF receptor superfamily. GITR, a co-stimulatory surface receptor, is activated upon binding to GITR Ligand (GITRL) and mediates co-stimulation of T-cell and NK responses and inhibits the suppressive activity of regulatory T-cells (Tregs). As such, GITR is an attractive immuno-oncology target for activation via GITR agonists. OMP-336B11 is a single-gene recombinant fusion protein consisting of two trimeric human GITRLs and a human immunoglobulin (IgG1) Fc domain. Murine preclinical studies using a surrogate GITRL-Fc fusion protein demonstrated robust antitumor efficacy. Functional characterization of OMP-336B11 in various human immune cell assays is presented here. In human peripheral blood mononuclear cells (PBMC), OMP-336B11 stimulated IL-2 cytokine release in a dose-dependent manner. In activated human T-cells, OMP-336B11 enhanced cell proliferation in a dose-responsive fashion. OMP-336B11 also augmented IL-2 induced IFNγ from human NK cells. To elicit NK mediated cytotoxicity of high GITR expressing cells (i.e., Tregs), OMP-336B11 is designed with an IgG1 Fc domain. Co-incubation of primary human NK cells (effector) and GITR expressing cells (target) resulted in an OMP-336B11 dependent dose-titratable increase in target cytotoxicity. Furthermore, OMP-336B11 agonistic activity was compared to anti-GITR agonist antibodies. By measuring cell proliferation and IFNγ from activated T-cells, OMP-336B11 demonstrated superior activity compared to the other agonist anti-GITR antibodies. In conclusion, OMP-336B11 is designed to induce effective GITR activation and also to mediate depletion of GITR-high cells. OMP-336B11 is currently undergoing phase I clinical study. Citation Format: Ivan H. Chan, Ming-Hong Xie, Andrew Lam, Fumiko T. Axelrod, Jennifer Elechko, Angie I. Park, Austin Gurney. In vitro functional activity of OMP-336B11, a GITRL-Fc fusion protein, on primary human immune cells [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 2726.

Abstract 3820: Novel PD-1/GITRL bispecific fusion protein delivering concurrent T cell co-stimulation and checkpoint pathway inhibition

Abstract Glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) is a co-stimulatory receptor, and programmed cell death protein 1 (PD-1) is an important co-inhibitory receptor involved in controlling T-cell activation and function. Preclinical studies using murine cross-reactive molecules demonstrate that the combination of an αPD-1 monoclonal antibody (mAb) with a GITR-ligand (GITRL) fusion protein can elicit synergistic antitumor activity and long-term survival benefit in syngeneic mouse tumor models. Here we describe the activity of two bispecific fusion proteins, each comprising two GITR ligand trimers fused to the Fc region of an αPD1 mAb, in an IgG1 (MEDI5771) or IgG4P (MEDI3387) format. We show that these bispecific molecules are able to concurrently engage both targets and are effective in cell-based PD-1 and GITR reporter assays, and in a range of human primary T-cell assays. Furthermore, the PD-1/GITRL FP bispecific constructs elicit T-cell responses in vitro that are at least equivalent to a combination of a) the parental molecules and b) MEDI1873 (a GITR agonist currently undergoing clinical evaluation) and an αPD-1 mAb. Moreover, using surrogate molecules we demonstrate that IV administration leads to dose-dependent pharmacodynamic responses on CD4+ and CD8+ T cells for both isotypes of the bispecific molecule. These data demonstrate that the antitumor activity for these novel molecules is equivalent to the combination of GITRL-FP and PD-1 mAb in the B16 model. In summary, the simultaneous dual targeting of PD-1 and GITR pathways mediated by these two novel bispecific molecules induces robust T-cell activity that is at least equivalent to a combination of molecules targeting these pathways, thereby demonstrating strong potential as a novel anticancer therapy. Citation Format: Simon J. Dovedi, Jonathan Seaman, Ines Osma-Garcia, Katharina Deschler, Edmund Poon, Ronald Herbst, Robert Wilkinson. Novel PD-1/GITRL bispecific fusion protein delivering concurrent T cell co-stimulation and checkpoint pathway inhibition [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 3820.

Changes in DNA Methylation of Glucocorticoid-Induced Tumor Necrosis Factor Receptor and Its Ligand in Liver Transplantation

Liver transplantation (LT) is the criterion standard of care in patients with end-stage liver disease and those with tumors of hepatic origin in the setting of liver dysfunction. Chronic immune rejection of the liver transplant can lead to bad prognosis for patients. Glucocorticoid-induced tumor necrosis factor receptor (GITR) play a key role in dominant immunologic self-tolerance maintained by CD25+/CD4+ regulatory T cells. Here, we investigated the DNA methylation variations of GITR and GITR ligand (GITRL) using pyrosequencing by analyzing blood DNA samples of patients after LT. Our results showed that the methylation level of certain CpGs, such as CpG_13, in GITRL was significantly reduced after LT. Furthermore, we found that the GITRL methylation statuses of cohorts with no chronic immune rejection were significantly lower compared with cohorts with chronic immune rejection after LT treatment. However, the methylation statuses of GITR were less varied. Using linear regression analysis, we further found that factors such as upper gastrointestinal hemorrhage, splenectomy, and creatinine might affect DNA methylation patterns in chronic immune rejection cohorts. These findings provide novel insights into the pivotal role of GITRL as a potential molecular biomarker for the triage of liver transplantation.

The Proinflammatory Cytokine GITRL Contributes to TRAIL-mediated Neurotoxicity in the HCN-2 Human Neuronal Cell Line.

Cytokines belonging to the TNF superfamily play a relevant role in neurodegenerative processes. Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL), released during neuronal injury, has proven to potently mediate and sustain neurotoxic processes leading to neuronal death. Similarly to TRAIL, the cytokine Glucocorticoid-induced TNF receptor ligand (GITRL) is able to transduce proapoptotic signals. In spite of the array of reports suggesting relationships between TRAIL and other cytokines, scanty data are, so far, available about a GITRL/TRAIL crosstalk. Here, we investigated possible interactions between TRAIL and the GITRL system in an in vitro model of neurodegeneration, using the human cortical neuronal cell line HCN-2. Cultured HCN-2 neurons were incubated at different times with GITRL and/or TRAIL, and thereafter nucleic acid and protein expression were measured. Real-time PCR analysis showed that the human cortical neuronal cell line HCN-2 does not express GITRL mRNA, but the latter is induced after treatment with TRAIL. In addition, HCN-2 cells did not express the GITRL receptor GITR mRNA, neither in control cultures, nor after treatment with TRAIL. All mRNA data were confirmed by western blot analysis of proteins. Cell viability assay showed that TRAIL, when associated to GITRL, was able to exert additive toxic effects. A counterproof was provided in experiments performed blocking GITRL, in which TRAIL-mediated toxicity appeared significantly reduced. Results suggest that GITRL/TRAIL redundancy during neurodegenerative processes implies extended potentiation of detrimental effects of both cytokines on neurons, eventually leading to larger cell damage and death. Finally, characterization of novel molecular targets within the TRAIL/GITRL interplay may represent a platform for innovative therapy of neurodegenerative disorders.