Glucocorticoid-induced Tumor Necrosis Factor Receptor Research Articles

Abstract 1795: Combination of cyclophosphamide chemotherapy with immune modulation overcomes resistance to checkpoint blockade in a pre-clinical melanoma model

Abstract Immune checkpoint blockade (ICB) with anti-CTLA-4 and anti-PD-1 is increasingly used to treat a variety of malignancies, but primary and acquired resistance limit the clinical benefit. Mechanism-guided approaches to overcome resistance and re-orchestrate an anti-tumor immune response could expand the clinical impact of ICBs. Cyclophosphamide (CTX) is an alkylating chemotherapeutic which is directly tumoricidal and also modulates immune response. CTX preferentially depletes certain T cell subsets (such as Tregs) and leads to homeostatic proliferation of antigen-specific T cells. We hypothesized that a dose of CTX prior to initiating ICB would counteract several known patterns of resistance and enhance anti-tumor response to ICB. The B16 murine melanoma model is largely refractory to inhibition of PD-1, CTLA-4, or combination CTLA-4+PD-1, all of which only modestly slow the growth of small tumors. One dose of CTX one day prior to each of these treatments significantly slows tumor growth and prolongs survival compared to CTX or ICB alone. This effect is most pronounced for the triple combination of CTX+ anti-CTLA-4+ anti-PD-1, where tumor regression was observed in 70% of mice and tumors were completely eradicated in 20%. In addition to standard ICB, we hypothesized that CTX would enhance response to immune modulatory molecules in clinical development. Glucocorticoid-induced TNFR family related gene (GITR) is a costimulatory molecule expressed primarily on the surface of T cell subsets. GITR engagement enhances activation, proliferation, and clonal expansion of effector T cells while hampering the suppressive function of Tregs. However, agonist anti-GITR antibodies have neither effectively controlled tumor growth when given as a monotherapy in preclinical models nor showed efficacy in clinical trials. In the B16 melanoma model, CTX + GITR engagement with the agonist monoclonal antibody DTA-1 slows tumor growth, elicits some tumor regression, and prolongs survival, whereas neither therapy alone controls tumor growth. Similar efficacy is observed in other tumor models (MCP-11 myeloma and CT26 colon carcinoma). Combination therapy with CTX + DTA-1 increases the ratio of CD8+ effector T cells to Tregs, at least in part by inducing Treg-specific activation-induced cell death. Single cell sequencing and flow cytometry demonstrated a marked increase in pro-inflammatory cytokines, cytolytic granules, and activation markers accompanied by reduced exhaustion markers in intratumoral CD8+ T cells. Overall, we demonstrate that a single dose of CTX added to standard and novel immune modulators is a potent combinatorial approach that primes an anti-tumor response, enhances T cell fitness, and overcomes primary resistance to ICB. The data presented here serve as the foundation for mechanistically-driven clinical trials in development. Citation Format: Allison Betof Warner, Daniel Hirschhorn, Levi M. Mangarin, Linda Hamadene, Adam D. Cohen, Gabrielle A. Rizzuto, Jedd D. Wolchok, Taha Merghoub. Combination of cyclophosphamide chemotherapy with immune modulation overcomes resistance to checkpoint blockade in a pre-clinical melanoma model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1795.

Emerging Targets of Immunotherapy in Gynecologic Cancer.

Although programmed cell death protein 1/programmed death-ligand 1 (PD-1/PD-L1) and cytotoxic T lymphocyte antigen-4 (CTLA-4) have been successfully applied in the treatment of tumors, their efficiency is still not high enough. New immune targets need to be identified in order to seek alternative treatment strategies for patients with refractory tumors. Immune targets can be divided into stimulating and inhibiting molecules according to their function after receptor–ligand binding. We herein present a compendious summary of emerging immune targets in gynecologic tumors. These targets included coinhibitory molecules, such as T cell immunoglobulin-3 (TIM-3), T cell immunoglobulin and ITIM domain (TIGIT), lymphocyte activation gene-3 (LAG-3), V-type immunoglobulin domain-containing suppressor of T cell activation (VISTA), and B7-H3 and B7-H4, and co-stimulatory molecules, such as CD27, OX40, 4–1BB, CD40, glucocorticoid-induced tumor necrosis factor receptor (GITR) and inducible co-stimulator (ICOS). In this review, the characteristics and preclinical/clinical progress of gynecological malignancies are briefly discussed. However, the potential mechanisms and interactions of immune targets need to be elucidated in further studies.

Phenotypic Changes of PD-1 and GITR in T Cells Are Associated With Hepatitis B Surface Antigen Seroclearance.

Regulatory T cells (Tregs) possess hepatitis B virus (HBV)-specific immunoregulatory effects in chronic HBV infection. The role of Tregs in spontaneous seroclearance of hepatitis B surface antigen (HBsAg) remains to be determined. We recruited treatment-naive chronic HBV patients achieving spontaneous HBsAg seroclearance (experimental group) and matched HBsAg-positive controls. Peripheral blood mononuclear cells were isolated using the Ficoll-Paque density gradient centrifugation method. The frequency of Tregs and inhibitory phenotypes and immunoregulatory cytokines of Tregs were detected by flow cytometry. Twenty-seven patients with HBsAg seroclearance (mean age: 52.40±6.00 y, 55.6% male) and 27 matched controls were recruited. Median HBsAg and HBV DNA levels in the control group were 2.80 (1.24 to 3.43) and 3.16 (1.68 to 3.85) log IU/mL, respectively. Mean frequencies of Tregs and expressions of FoxP3+ Tregs were comparable in both groups (both P>0.05). The mean expression of programmed death 1 (PD-1) and glucocorticoid-induced TNFR family-related gene (GITR) in total CD4+ T cells were significantly downregulated in the experimental group when compared with the control group (10.62% vs. 13.85%, P=0.003; 16.20% vs. 27.02%, P=0.002, respectively). When compared with the control group, PD-1+CD4+ Tregs expression in the experimental group was significantly downregulated (13.85% vs. 10.62%, P=0.003). A similar phenomenon was noted for GITR+CD8+ Tregs (20.16% vs. 14.08%, P=0.049). Intracellular cytokine productions showed no significant differences (all P>0.05). The reduced expression of PD-1 and GITR might attenuate the immunosuppressive capability of Tregs. Decreased expression on CD4+ T cells might represent an enhanced antiviral function, playing a role in initiating the "functional cure" of chronic HBV infection.

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.

Identification of a Novel Antisepsis Pathway: Sectm1a Enhances Macrophage Phagocytosis of Bacteria through Activating GITR.

The inability to effectively control invading bacteria or other pathogens is a major cause of multiple organ dysfunction and death in sepsis. As the first-line defense of the immune system, macrophages play a crucial role in the removal of pathogens during sepsis. In this study, we define secreted and transmembrane 1A (Sectm1a) as a novel ligand of glucocorticoid-induced TNFR (GITR) that greatly boosts macrophage phagocytosis and bactericidal capacity. Using a global Sectm1a knockout (KO) mouse model, we observed that Sectm1a deficiency significantly suppressed phagocytosis and bactericidal activity in both recruited macrophages and tissue-resident macrophages, which consequently aggravated bacterial burden in the blood and multiple organs and further increased systemic inflammation, leading to multiple organ injury and increased mortality during polymicrobial sepsis. By contrast, treatment of septic mice with recombinant Sectm1a protein (rSectm1a) not only promoted macrophage phagocytosis and bactericidal activity but also significantly improved survival outcome. Mechanistically, we identified that Sectm1a could bind to GITR in the surface of macrophages and thereby activate its downstream PI3K-Akt pathway. Accordingly, rSectm1a-mediated phagocytosis and bacterial killing were abolished in macrophages by either KO of GITR or pharmacological inhibition of the PI3K-Akt pathway. In addition, rSectm1a-induced therapeutic effects on sepsis injury were negated in GITR KO mice. Taken together, these results uncover that Sectm1a may represent a novel target for drug development to control bacterial dissemination during sepsis or other infectious diseases.

GITR controls intestinal inflammation by suppressing IL-15-dependent NK cell activity.

Glucocorticoid-induced TNFR family related gene (GITR) is a member of the TNFR superfamily that is expressed on cells of the immune system. Although the protective and pathogenic roles of GITR in T cell immunity are well characterized, the role of GITR in innate immunity in the intestinal tissues has not been well clarified. In this study, using a dextran sulfate sodium (DSS)-induced colitis model in mice, we found that GITR-deficiency rendered mice more susceptible to acute intestinal inflammation and that a significantly higher number of activated natural killer (NK) cells was accumulated in the colonic lamina propria of Gitr-/- mice as compared to wild-type mice. Additionally, Rag2-/- Gitr-/- mice, which lack T cells but have NK cells, also displayed more severe colonic inflammation than Rag2-/- mice. In contrast, an anti-GITR agonistic antibody significantly alleviated colitis in Rag2-/- mice. Engagement of GITR inhibited IL-15-mediated activating signaling events in NK cells, which include cell activation and proliferation, and production of cytokines and cytotoxic granules. Taken together, our results provide the first evidence that GITR negatively controls intestinal inflammation through NK cell functions.

Glucocorticoid-induced tumour necrosis factor receptor family-related protein (GITR) drives atherosclerosis in mice and is associated with an unstable plaque phenotype and cerebrovascular events in humans.

GITR-a co-stimulatory immune checkpoint protein-is known for both its activating and regulating effects on T-cells. As atherosclerosis bears features of chronic inflammation and autoimmunity, we investigated the relevance of GITR in cardiovascular disease (CVD). GITR expression was elevated in carotid endarterectomy specimens obtained from patients with cerebrovascular events (n = 100) compared to asymptomatic patients (n = 93) and correlated with parameters of plaque vulnerability, including plaque macrophage, lipid and glycophorin A content, and levels of interleukin (IL)-6, IL-12, and C-C-chemokine ligand 2. Soluble GITR levels were elevated in plasma from subjects with CVD compared to healthy controls. Plaque area in 28-week-old Gitr-/-Apoe-/- mice was reduced, and plaques had a favourable phenotype with less macrophages, a smaller necrotic core and a thicker fibrous cap. GITR deficiency did not affect the lymphoid population. RNA sequencing of Gitr-/-Apoe-/- and Apoe-/- monocytes and macrophages revealed altered pathways of cell migration, activation, and mitochondrial function. Indeed, Gitr-/-Apoe-/- monocytes displayed decreased integrin levels, reduced recruitment to endothelium, and produced less reactive oxygen species. Likewise, GITR-deficient macrophages produced less cytokines and had a reduced migratory capacity. Our data reveal a novel role for the immune checkpoint GITR in driving myeloid cell recruitment and activation in atherosclerosis, thereby inducing plaque growth and vulnerability. In humans, elevated GITR expression in carotid plaques is associated with a vulnerable plaque phenotype and adverse cerebrovascular events. GITR has the potential to become a novel therapeutic target in atherosclerosis as it reduces myeloid cell recruitment to the arterial wall and impedes atherosclerosis progression.

GITR Agonism Triggers Antitumor Immune Responses through IL21-Expressing Follicular Helper T Cells.

Although treatment with the glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) agonistic antibody (DTA-1) has shown antitumor activity in various tumor models, the underlying mechanism is not fully understood. Here, we demonstrate that interleukin (IL)-21-producing follicular helper T (Tfh) cells play a crucial role in DTA-1-induced tumor inhibition. The administration of DTA-1 increased IL21 expression by Tfh cells in an antigen-specific manner, and this activation led to enhanced antitumor cytotoxic T lymphocyte (CTL) activity. Mice treated with an antibody that neutralizes the IL21 receptor exhibited decreased antitumor activity when treated with DTA-1. Tumor growth inhibition by DTA-1 was abrogated in Bcl6 fl/fl Cd4 Cre mice, which are genetically deficient in Tfh cells. IL4 was required for optimal induction of IL21-expressing Tfh cells by GITR costimulation, and c-Maf mediated this pathway. Thus, our findings identify GITR costimulation as an inducer of IL21-expressing Tfh cells and provide a mechanism for the antitumor activity of GITR agonism.

Evaluation of glucocorticoid-induced TNF receptor (GITR) expression in breast cancer and across multiple tumor types.

Glucocorticoid-induced TNF receptor (GITR) is an emerging immunotherapy target that is expressed at high levels on regulatory T cells. Agonistic anti-GITR antibodies have anti-tumor activity in cancer mouse models, and recent phase 1 trials have demonstrated their safe pharmacological profile. However, there is limited knowledge on the relationship between GITR expression and the tumor microenvironment. GITR protein expression was assayed by immunohistochemistry on 3992 breast cancer surgical excision specimens assembled into tissue microarrays and scored visually by a pathologist for GITR expression on tumor-infiltrating lymphocytes and on carcinoma cells. GITR expression by the malignant cells was further surveyed in gastrointestinal stromal tumor (N = 713), lung carcinoma (N = 705), pancreatic cancer (N = 486), ovarian cancer (N = 445), bladder cancer (N = 88), prostate cancer (N = 88), testicular cancer (N = 76), melanoma (N = 75), renal cell carcinoma (N = 68), epithelioid sarcoma (N = 53), and neuroendocrine tumors (N = 41). In breast cancer, GITR expression on tumor-infiltrating lymphocytes (12.4%) correlated with other immune response biomarkers (PD-L1+ on tumor cells, and PD-1+, LAG-3+, TIM-3+ lymphocytes; p < 0.001), and T-cell markers (CD8+, FOXP3+; p < 0.001). GITR+ carcinoma cells were observed in 6.0% of breast cancer cases and correlated with worse relapse-free survival (p = 0.015). Among the additional tumor types examined, cancers with GITR+ malignant cells included bladder cancer (5.7%), primary (but not metastatic) melanoma (4.5%), and ovarian cancer (3.2%); no expression was identified among examined sarcomas. To our knowledge, this is the first immunohistochemistry study to report the frequency and pattern of GITR expression in a large breast cancer cohort, or to report membranous GITR expression on malignant cells. The co-infiltration of GITR with other immune biomarkers and T-cell markers supports a potential role for anti-GITR agents in combination immunotherapies. In addition, GITR expression on carcinoma cells could imply the existence of a novel cancer immune evasion strategy worthy of further investigation.

GITR shapes humoral immunity by controlling the balance between follicular T helper cells and regulatory T follicular cells

Follicular helper CD4+ T-cells (Tfh) control humoral immunity by driving affinity maturation and isotype-switching of activated B-cells. Tfh localize within B-cell follicles and, upon encounter with cognate antigen, drive B-cell selection in germinal centers (GCs) as GC-Tfh. Tfh functionality is controlled by Foxp3-expressing Tfh, which are known as regulatory T follicular cells (Tfr). Thus far, it remains unclear which factors determine the balance between these functionally opposing follicular T-cell subsets. Here, we demonstrate in human and mouse that Tfh and GC-Tfh, as well as their regulatory counterparts, express glucocorticoid-induced TNF receptor related protein (GITR) on their surface. This costimulatory molecule not only helps to identify follicular T-cell subsets, but also increases the ratio of Tfh vs. Tfr, both within and outside the GC. Correspondingly, GITR triggering increases the number of IL-21 producing CD4+ T-cells, which also produce more IFN-γ and IL-10. The latter are known switch factors for IgG2c and IgG1, respectively, which corresponds to a concomitant increase in IgG2c and IgG1 production upon GITR-mediated costimulation. These results demonstrate that GITR can skew the functional balance between Tfh and Tfr, which offers new therapeutic possibilities in steering humoral immunity.

Biological role of GITR/GITRL in attributes and immune responses of macrophage.

Glucocorticoid-induced tumor necrosis factor receptor family-related protein ligand (GITRL), a member of the tumor necrosis factor superfamily, is expressed in APCs and acts as a costimulatory molecule in the immune system. Although the glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR)/GITRL system has been modulated to promote or decrease T cell-related responses in multiple diseases, studies in macrophages are limited. To address this issue, we compared the expression of GITRL in various types of macrophages and analyzed whether GITRL can affect the fundamental properties and major functions of these cells. Our results demonstrated that M1 polarized macrophages had the highest GITRL levels. Furthermore, GITRL overexpression skewed macrophage polarization toward the M1 phenotype, accelerating proliferation and migration and regulating phagocytosis and killing function. Moreover, GITRL-silenced cells showed a loss of these functions, further confirming its vital role. We also developed an acute peritonitis mouse model, in which macrophages were driven to differentiate into a proinflammatory phenotype with GITRL up-regulation, triggering a positive feedback loop. Our results provide molecular insight into how the GITR/GITRL system modulates innate immune responses, suggesting that manipulation of the GITR/GITRL system to treat diseases depends not only on T cell regulation but also on macrophage participation.

A personalised approach for anti-GITR-based immunotherapy in pre-clinical models of pancreatic ductal adenocarcinoma

Abstract Background Patient outcomes with pancreatic ductal adenocarcinoma (PDA) remain dismal, despite the introduction of combination chemotherapies. Response to therapy is hampered by tumour stroma, which is highly heterogeneous but poorly characterised. The clinical success of immunotherapy in certain cancer types justifies investigation in PDA using a personalised approach of selecting pre-clinical models. Methods Patient PDA samples (n = 70; International Cancer Genome Consortium) were subjected to hierarchical clustering using a gene set representing CD8 and regulatory T cells to identify sub-groups. Syngeneic mouse models recapitulating these sub-groups (cross-species analysis) were developed using cell lines from genetically engineered mouse (GEM) models. Models bearing subcutaneous tumours were treated with anti-GITR agonist (DTA-1; 10mg/kg) or isotype (IgG2b) and tissues were collected for 12-color fluorescence activated cell sorting (FACS), NanoString and immunohistochemistry (IHC). Results Using clustering analysis, we identified four distinct patient PDA immune sub-groups with differential expression of T cell genes. These sub-groups showed differential expression of glucocorticoid-induced TNF receptor (GITR) and its associated genes. Hence, we hypothesised that these groups may respond differently to anti-GITR therapy in vivo. As expected, the three syngeneic models showed differential response to anti-GITR treatment. Model with increased GITR expression (>2-fold; NanoString and immunoblot) showed a significant (p = 0.02) survival benefit and a modest decrease in tumour burden upon anti-GITR treatment (n = 10) compared to the isotype control (n = 7) and the other two models. We observed anti-GITR mediated increase in GITR+ CD8+ cells and no change in GITR+ FOXP3+ cells (FACS, NanoString and IHC) along with increased gene expression of Pdcd1, Lag3, Spn and Itk. Conclusion Our models recapitulate the immunological gene expression profiles observed in patient PDA samples making them a highly useful resource to study personalized immunotherapies. This integrated study highlights the importance of patient selection to maximize therapy benefit and spare immune related adverse events. Legal entity responsible for the study The authors. Funding Bristol-Myers Squibb. Disclosure D. Cunningham: Research grant / Funding (self): Amgen; Research grant / Funding (self): Sanofi; Research grant / Funding (self): Merrimack; Research grant / Funding (self): AstraZeneca; Research grant / Funding (self): Celgene; Research grant / Funding (self): MedImmune; Research grant / Funding (self): Bayer; Research grant / Funding (self): 4SC; Research grant / Funding (self): Clovis; Research grant / Funding (self): Eli Lilly; Research grant / Funding (self): Janssen; Research grant / Funding (self): Merck. N. Starling: Research grant / Funding (self): AstraZeneca; Research grant / Funding (self): Bristol-Myers Squibb; Research grant / Funding (self): Pfizer; Travel / Accommodation / Expenses: AstraZeneca; Travel / Accommodation / Expenses: Bristol-Myers Squibb; Travel / Accommodation / Expenses: Eli Lilly; Travel / Accommodation / Expenses: Merck; Travel / Accommodation / Expenses: Roche; Honoraria (self): Eli Lilly; Honoraria (self): Merck Serono; Honoraria (self): MSD Oncology; Honoraria (self): Pierre Fabre; Advisory / Consultancy: Pfizer; Advisory / Consultancy: AstraZeneca; Advisory / Consultancy: Servier. A. Sadanandam: Research grant / Funding (self): Bristol-Myers Squibb; Research grant / Funding (self): Merck KGaa; Research grant / Funding (self): Pierre Fabre. All other authors have declared no conflicts of interest.

Next generation of anti-immune checkpoints antibodies

Immune checkpoints balance initial antigen-driven T cell stimulation by enhancing or dampening activation, allowing co-existence of efficient immune responses and maintenance of self-tolerance. In oncology, checkpoints currently targeted by inhibitors to amplify activity of T cell, NK cells or myeloid cells responses comprise CTLA-4 (cytolytic T-lymphocyte-associated antigen 4 or CD152), PD-1 (programmed cell death 1, or CD279), PD-L1 ( programmed cell death-ligand 1, or CD274), LAG-3 (Lymphocyte-activation gene 3, or CD223), TIM3 (T-cell immunoglobulin and mucin-domain containing-3), TIGIT (T cell immunoreceptor with Ig and ITIM domains ), VISTA (V-domain Ig suppressor of T cell activation), B7/H3 (or CD276), KIR (killer-cell immunoglobulin-like receptor), NKG2A, CD39, CD73, CSF1R (colony-stimulating factor 1 receptor), CD47 or CD172a. Other "checkpoints" are being pharmacologically triggered in order to directly amplify T cell co-stimulation. Among these molecules, CD28, CD137 (also called 4-1BB), OX40 [also called tumor necrosis factor receptor superfamily, member 4 (TNFRSF4)], GITR (Glucocorticoid-induced tumor necrosis factor receptor family-related protein) or CD40 are also tested in oncology, most often in combination with an inhibitory checkpoint inhibitor. In autoimmune and inflammatory diseases, checkpoint inhibitors or activators (LAG-3, CD28, CD40L) are also being tested. In this review, we focus on some modulators of immune checkpoints for which the mechanism of action has been particularly studied. As this description cannot be exhaustive, we have grouped in Table I all monoclonal antibodies (MAbs) or recombinant proteins in clinical use (to our knowledge), modulating the action of a control point of the immune system.

A Real-time Potency Assay for Chimeric Antigen Receptor T Cells Targeting Solid and Hematological Cancer Cells.

Chimeric antigen receptor (CAR) T-cell therapy for cancer has achieved significant clinical benefit for resistant and refractory hematological malignancies such as childhood acute lymphocytic leukemia. Efforts are currently underway to extend this promising therapy to solid tumors in addition to other hematological cancers. Here, we describe the development and production of potent CAR T cells targeting antigens with unique or preferential expression on solid and liquid tumor cells. The in vitro potency of these CAR T cells is then evaluated in real-time using the highly sensitive impedance-based xCELLigence assay. Specifically, the impact of different costimulatory signaling domains, such as glucocorticoid-induced tumor necrosis factor receptor (TNFR)-related protein (GITR), on the in vitro potency of CAR T cells is examined. This report includes protocols for: generating CAR T cells for preclinical studies using lentiviral gene transduction, expanding CAR T cells, validating CAR expression, and running and analyzing xCELLigence potency assays.

Safety, Tolerability, and Potential Clinical Activity of a Glucocorticoid-Induced TNF Receptor–Related Protein Agonist Alone or in Combination With Nivolumab for Patients With Advanced Solid Tumors

Multiple immunostimulatory agonist antibodies have been clinically tested in solid tumors to evaluate the role of targeting glucocorticoid-induced tumor necrosis factor (TNF) receptor-related protein in anticancer treatments. To evaluate the safety and activity of the fully human glucocorticoid-induced TNF receptor-related protein agonist IgG1 monoclonal antibody BMS-986156 with or without nivolumab in patients with advanced solid tumors. This global, open-label, phase 1/2a study of BMS-986156 with or without nivolumab enrolled 292 patients 18 years or older with advanced solid tumors and an Eastern Cooperative Oncology Group performance status of 1 or less. Prior checkpoint inhibitor therapy was allowed. Monotherapy and combination dose-escalation cohorts ran concurrently to guide expansion doses beginning October 16, 2015; the study is ongoing. The protein agonist BMS-986156 was administered intravenously at a dose of 10, 30, 100, 240, or 800 mg every 2 weeks as monotherapy, and in the combination group 30, 100, 240, or 800 mg plus 240 mg of nivolumab every 2 weeks; same-dose cohorts were pooled for analysis. One cohort also received 480 mg of BMS-986156 plus 480 mg of nivolumab every 4 weeks. The primary end points were safety, tolerability, and dose-limiting toxic effects. Additional end points included antitumor activity per Response Evaluation Criteria in Solid Tumors, version 1.1, and exploratory biomarker analyses. With a follow-up range of 1.4 to 101.7 weeks (follow-up ongoing), 34 patients (16 women and 18 men; median age, 56.6 years [range, 28-75 years]) received monotherapy (4 patients completed initial treatment), and 258 patients (140 women and 118 men; median age, 60 years [range, 21-87 years]) received combination therapy (65 patients completed initial treatment). No grade 3 to 5 treatment-related adverse events occurred with BMS-986156 monotherapy; grade 3 to 4 treatment-related adverse events occurred in 24 patients (9.3%) receiving BMS-986156 plus nivolumab, with no grade 5 treatment-related adverse events. One dose-limiting toxic effect (grade 4 elevated creatine phosphokinase levels) occurred in a patient receiving 800 mg of BMS-986156 plus 240 mg of nivolumab every 2 weeks; BMS-986156 with or without nivolumab exhibited linear pharmacokinetics with dose-related increase after a single dose. Peripheral T-cell and natural killer-cell proliferation increased after administration of BMS-986156 with or without nivolumab. No consistent and significant modulation of intratumoral CD8+ T cells and FoxP3+ regulatory T cells was observed. No responses were seen with BMS-986156 alone; objective response rates ranged from 0% to 11.1% (1 of 9) across combination therapy cohorts, with a few responses observed in patients previously treated with anti-programmed death receptor (ligand) 1 therapy. Based on this cohort, BMS-986156 appears to have had a manageable safety profile, and BMS-986156 plus nivolumab demonstrated safety and efficacy comparable to historical data reported for nivolumab monotherapy. ClinicalTrials.gov identifier: NCT02598960.

From the Editor’s Desk…: October 2019

From the Editor’s Desk…: October 2019

Abstract 3204: Pre-immunization of donor lymphocytes with GITR agonistic antibody enhances antitumor immunity in autologous hematopoietic stem cell transplantation

Abstract Lymphopenia-induced homeostatic proliferation (HP) of T cells following autologous hematopoietic stem cell transplantation (HSCT) skews the T-cell repertoire by engaging tumor-associated antigens, and induces an antitumor immunity. However, the cure by autologous HSCT alone is difficult in case of solid cancers. Glucocorticoid-induced tumor necrosis factor receptor (GITR) is well-known immune-checkpoint molecules. GITR-GITR ligand interaction can provide a co-stimulatory signal to both CD4+ and CD8+ naïve T cells, enhancing proliferation and effector function. Since the tumor-reactive lymphocytes preferentially proliferate during the condition of HP, due to the synergic effect with encountering their cognate antigens in tumor-bearing host, we hypothesize that administration of GITR agonistic antibody (Ab) could increase tumor-responsive T cells in the graft and induce a strong antitumor immunity in HSCT recipients. First, we confirmed that the intraperitoneal administration of GITR Ab into CT26 subcutaneous tumor model mice significantly increased the number of IFN-γ+ lymphocytes in response to CT26 cells in the spleens and regional lymph nodes. Second, to examine the antitumor effect of HSCT, the bone marrow cells and lymphocytes were harvested from CT26 tumor-bearing mice as donor cells, and infused into the lethally-irradiated recipient mice with CT26 inoculation (autologous HSCT model). In the HSCT recipients infused with primed lymphocytes by GITR Ab, the CT26 tumor growth was markedly suppressed compared with the recipients infused with non-primed lymphocytes, and tumors disappeared in all recipient mice infused with primed lymphocytes. An ELISpot assay showed that the number of IFN-γ+ spots in response to CT26 cells was significantly increased in HSCT recipient mice infused with GITR Ab-primed and non-primed donor lymphocytes as compared to wild type mice until 4 weeks after HSCT; while the frequency in recipients with primed lymphocytes was markedly elevated compared with that in mice with non-primed lymphocytes at 2 weeks after HSCT. At present, autologous HSCT is clinically practiced after intensive chemotherapy in patients with lymphomas and solid cancers such as neuroblastoma and sarcoma, the infusion of hematopoietic stem cells and pre-immunized lymphocytes after high dose chemotherapy is an attractive and feasible clinical introduction of HSCT-mediated immunotherapy. Although the subcutaneous tumors of NHOS murine osteosarcoma cells were resistant for HSCT, the infusion of GITR Ab-primed lymphocytes significantly induced stronger antitumor effect than the non-primed lymphocytes did. In conclusion, the combination of HSCT with pre-immunization by GITR Ab can induce a strong antitumor immunity. This therapeutic strategy deserves an evaluation in future clinical trial for solid cancers. Citation Format: Kenta Narumi, Marina Henmi, Chihiro Shibasaki, Aya Hirata, Yukihiro Mizoguchi, Kazunori Aoki. Pre-immunization of donor lymphocytes with GITR agonistic antibody enhances antitumor immunity in autologous hematopoietic stem cell transplantation [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 3204.

1748-P: Brucine Topical Administration Promotes Wound Healing through Downregulation of Proinflammatory Markers and Chemokine Expressions in Alloxan-Induced Diabetic Mice

Diabetes mellitus is a complex metabolic disease and delayed wound healing constitutes one of the most serious diabetes-associated complications. Diabetes can impede the wound healing process by inducing inflammation which can lead to delayed maturation of granulation tissues and reduced wound parallel tension. Brucine, one of the major constituents in Strychnos nux-vomica has antioxidant and anti-inflammatory properties. In this study, we investigated the effects of topical administration of brucine on the key mediators of wound healing, namely T cell subsets, glucocorticoid-induced tumor necrosis factor receptor expressing cells, CD4+CD25+Foxp3+ regulatory T (Treg) cells, Th17 cells, Th1 cytokines, and inflammatory mediator mRNA expression.In current study, 5 groups of mice were used (10/group): group 1, the nondiabetic normal control; group 2, wound in nondiabetic mice; group 3, wound in diabetic mice; group 4, brucine 20mg/kg treated wounds in diabetic mice. Wound size was recorded on every third day and after 14 days of treatment, the heparinized whole blood and the wound tissue of all the groups was collected and tested. Brucine administration showed a significant reduction in wound size, the levels of GITR-expressing cells, and Th1 cytokines as well as substantial down regulation of mRNA expression of the inflammatory mediators compared with the diabetic mice. Brucine also significantly up regulated the number of Tregs or also induced Th17/Treg balance and modulated various pro-inflammatory and anti-inflammatory cytokines and the mRNA expression of their mediators. Furthermore, histopathological examination showed complete re-epithelization, decreased inflammatory cells and presence of granulation tissue in the brucine treated mice. Therefore, our results suggest the beneficial effect of brucine in enhanced wound healing and rebalancing the wound environment in diabetes. Disclosure H. Madkhali: None. M. Ganaie: None. M.N. Ansari: None.

Soluble glucocorticoid-induced tumor necrosis factor receptor regulates Helios expression in myasthenia gravis

BackgroundHelios is important for functional and phenotype stability of regulatory T cells (Tregs). However, the role of Helios in autoimmune diseases and its regulation remains unclear. This study aimed to investigate the role of Helios+ Tregs in myasthenia gravis (MG) and glucocorticoid-induced tumor necrosis factor receptor (GITR) and its ligand (GITRL) in the modulation of Helios.MethodMulticolor flow cytometry was performed to analyze Helios+ Tregs in peripheral blood from MG patients and healthy donors (HDs). Enzyme-linked immunosorbent assay (ELISA) was used to determine the levels of soluble GITRL/GITR in plasma. Tregs were isolated via magnetic separation and treated with recombinant GITRL and GITR-Fc. Membrane GITRL on Tregs and expression of Helios and other markers (FOXP3, CD25, CD39, CTLA-4, PD-L1 and IL-10) involved in immunosuppressive activity were determined by flow cytometry.ResultBoth Helios+ Tregs and soluble GITR were decreased in generalized MG (GMG) patients (n = 14), compared with HDs (n = 14) and ocular MG (OMG) patients (n = 16). Helios+ Tregs possessed greater immunosuppressive capacity compared to Helios− Tregs. Further analysis indicates soluble GITR was negatively correlated with quantitative MG score and promoted Helios expression and enhanced function of Tregs independently of membrane GITRL.ConclusionThis work demonstrates abnormal changes in Helios+ Tregs and soluble GITR in MG, as well as direct regulation of Helios by GITR in the context of Tregs. This work provides new insight into the role of GITR in the regulatory pathway of Helios and pathogenesis of MG.