Inhibitory TIM-3 Research Articles

Regulatory and memory T lymphocytes infiltrating prostate tumors predict long term clinical outcomes.

The localization, density but mostly the phenotype of tumor infiltrating lymphocytes (TIL) provide important information on the initial interaction between the host immune system and the tumor. Our objective was to assess the prognostic significance of T (CD3+), T regulatory (Treg) (FoxP3+) and T memory (Tmem) (CD45RO+) infiltrating lymphocytes and of genes associated with TIL in prostate cancer (PCa). Immunohistochemistry (IHC) was used to assess the infiltration of CD3+, FoxP3+ and CD45RO+ cells in the tumor area, tumor margin and adjacent normal-like epithelium of a series of 98 PCa samples with long clinical follow-up. Expression of a panel of 31 TIL-associated genes was analyzed by Taqman Low-Density Array (TLDA) technology in another series of 50 tumors with long clinical follow-up. Kaplan-Meier and Cox proportional hazards regression analyses were performed to determine association of these markers with biochemical recurrence (BCR), need for definitive androgen deprivation therapy (ADT) or lethal PCa. TIL subtypes were present at different densities in the tumor, tumor margin and adjacent normal-like epithelium, but their density and phenotype in the tumor area were the most predictive of clinical outcomes. In multivariate analyses, a high density of Treg (high FoxP3+/CD3+ cell ratio) predicted a higher risk for need of definitive ADT (HR=7.69, p=0.001) and lethal PCa (HR=4.37, p=0.04). Conversely, a high density of Tmem (high CD45RO+/CD3+ cell ratio) predicted a reduced risk of lethal PCa (HR=0.06, p=0.04). TLDA analyses showed that a high expression of FoxP3 was associated with a higher risk of lethal PCa (HR=5.26, p=0.02). Expression of CTLA-4, PD-1, TIM-3 and LAG-3 were correlated with that of FoxP3. Amongst these, only a high expression of TIM-3 was associated with a significant higher risk for definitive ADT in univariate Cox regression analysis (HR=3.11, p=0.01). These results show that the proportion of Treg and Tmem found within the tumor area is a strong and independent predictor of late systemic progression of PCa. Our results also suggest that inhibition of TIM-3 might be a potential approach to counter the immunosuppressive functions of Treg in order to improve the anti-tumor immune response against PCa.

TIM-3 Expression on Dendritic Cells in Colorectal Cancer.

TIM-3 was originally identified as a negative regulator of helper T cells and is expressed on dendritic cells (DCs). Since the inhibition of TIM-3 on DCs has been suggested to enhance T cell-mediated anti-tumor immunity, we examined its expression on DCs within the tumor microenvironment (TME) in colorectal cancer (CRC) using transcriptomic data from a public database (n = 592) and immunohistochemical evaluations from our cohorts of CRC (n = 115). The expression of TIM-3 on DCs in vitro was examined by flow cytometry, while the expression of its related molecules, cGAS and STING, on immature and mature DCs was assessed by Western blotting. The expression of HAVCR2 (TIM-3) was strongly associated with the infiltration of DCs within the TME of CRC. Immunohistochemical staining of clinical tissue samples revealed that tumor-infiltrating DCs expressed TIM-3; however, their number at the tumor-invasive front significantly decreased with stage progression. TIM-3 expression was higher on immature DCs than on mature DCs from several different donors (n = 6). Western blot analyses showed that the expression of STING was higher on mature DCs than on immature DCs, which was opposite to that of TIM-3. We demonstrated that TIM-3 was highly expressed on tumor-infiltrating DCs of CRC and that its expression was higher on immature DCs than on mature DCs.

TIM-3 blockade in diffuse intrinsic pontine glioma models promotes tumor regression and antitumor immune memory

Diffuse intrinsic pontine glioma (DIPG) is an aggressive brain stem tumor and the leading cause of pediatric cancer-related death. To date, these tumors remain incurable, underscoring the need for efficacious therapies. In this study, we demonstrate that the immune checkpoint TIM-3 (HAVCR2) is highly expressed in both tumor cells and microenvironmental cells, mainly microglia and macrophages, in DIPG. We show that inhibition of TIM-3 in syngeneic models of DIPG prolongs survival and produces long-term survivors free of disease that harbor immune memory. This antitumor effect is driven by the direct effect of TIM-3 inhibition in tumor cells, the coordinated action of several immune cell populations, and the secretion of chemokines/cytokines that create a proinflammatory tumor microenvironment favoring a potent antitumor immune response. This work uncovers TIM-3 as a bona fide target in DIPG and supports its clinical translation.

Discovery of Small-Molecule TIM-3 Inhibitors for Acute Myeloid Leukemia Using Pharmacophore-Based Virtual Screening.

T-cell immunoglobulin and mucin domain 3 (TIM-3) is a negative immune checkpoint that represents a promising target for cancer immunotherapy. Although encouraging results have been observed for TIM-3 inhibition in the context of acute myeloid leukemia (AML), targeting TIM-3 is currently restricted to monoclonal antibodies (mAbs). To fill this gap, we implemented a pharmacophore-based screening approach to identify small-molecule TIM-3 inhibitors. Our approach resulted in the identification of hit compounds with TIM-3 binding affinity. Subsequently, we used the structure-activity relationship (SAR) by a catalog approach to identify compound A-41 with submicromolar TIM-3 binding affinity. Remarkably, A-41 demonstrated the ability to block TIM-3 interactions with key ligands and inhibited the immunosuppressive function of TIM-3 using an in vitro coculture assay. This work will pave the way for future drug discovery efforts aiming at the development of small-molecule inhibitors TIM-3 for AML.

Abstract 6369: Modulation of tumor microenvironment with TIM-3 blockade improves survival in diffuse midline glioma models

Abstract Purpose of Study: Diffuse midline glioma (DMG) is an aggressive brain tumor and the leading cause of pediatric death caused by cancer. Despite great strides in the understanding of this disease, survival is still dismal. One of the objectives of our lab is to modulate the tumor microenvironment (TME) towards a proinflammatory phenotype to render these tumors amenable to immunotherapy. TIM-3 is a member of the TIM family of immunoregulatory proteins expressed on multiple immune cell types, including T-cells, NK, myeloid populations, and microglia, regulating adaptive and innate immunity. Therefore, the aim of this project is to study the antitumor effect of the anti-TIM-3 monoclonal antibody and its effect on the DMG tumor microenvironment. Experimental Procedures: TIM-3 expression in DMG patients was analyzed using total mRNA sequencing data and single-cell RNAseq data. To perform all the experiments, DIPG murine cell lines were used (NP53 and XFM). For in vivo experiments, cells were injected into the pons of mice using a screw-guided system. The antibody was administered intracranially (25µg) with the same system and two times intraperitoneally (10mg/kg) 3, 7, and 11 days after the cell implantation respectively. Tumor immune populations, chemokines, and cytokines were analyzed by flow cytometry. Results: In silico assessment of TIM-3 expression in DMG mRNA and single-cell datasets showed a robust expression of this gene mainly in microglia and macrophages uncovering this molecule as a potential target in DMGs. In vivo studies showed that TIM-3 blockade with an antibody significantly increased the overall survival of two DMG immunocompetent orthotopic models, led to long-term survivors (50%), and showed immune memory. TIM-3 inhibition resulted in significant increase in the number and proliferative state of microglia, NK, and CD8+ cells and higher levels of IFNγ, GrzB and TNFα corresponding to NK and T-cell activate phenotypes. Interestingly, there was a decrease in the Treg population, which caused an increase in the pro-inflammatory CD8/Treg ratio. Chemokine studies demonstrated an augmentation of CCL5, CCL2 chemotactic chemokines, and CXCL10, IL-1β and IFN-γ pro-inflammatory cytokines in the tumor microenvironment of treated mice. Additionally, DCs, CD4+, and CD8+ cells were increased in treated draining lymph nodes and of functional significance, expressed higher amounts of pro-inflammatory cytokines than in control mice. Interestingly, the depletion NK, CD4 and CD8 immune populations did not completely abrogate the treatment efficacy. However, microglia and macrophages depletion with an anti-CSF1R resulted in a total loss of efficacy indicating a critical role of these populations in the effect of TIM-3 blockade. Conclusions: These data uncover TIM-3 as a potential target for the treatment of DMG and its role as an immune regulator of the DMG tumor microenvironment. Citation Format: Iker Ausejo-Mauleon, Sara Labiano, Virginia Laspidea, Daniel de la Nava, Oren Becher, Mariella G Filbin, Fernando Pastor, Marta M Alonso. Modulation of tumor microenvironment with TIM-3 blockade improves survival in diffuse midline glioma models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6369.

Combined Immune Checkpoint Blockade Enhances Antiviral Immunity against Bovine Leukemia Virus.

Bovine leukemia virus (BLV) is a retrovirus that causes enzootic bovine leukosis (EBL) in cattle and is widespread in many countries, including Japan. Recent studies have revealed that the expression of immunoinhibitory molecules, such as programmed death-1 (PD-1) and PD-ligand 1, plays a critical role in immunosuppression and disease progression during BLV infection. In addition, a preliminary study has suggested that another immunoinhibitory molecule, T-cell immunoglobulin domain and mucin domain-3 (TIM-3), is involved in immunosuppression during BLV infection. Therefore, this study was designed to further elucidate the immunoinhibitory role of immune checkpoint molecules in BLV infection. TIM-3 expression was upregulated on peripheral CD4+ and CD8+ T cells in BLV-infected cattle. Interestingly, in EBL cattle, CD4+ and CD8+ T cells infiltrating lymphomas expressed TIM-3. TIM-3 and PD-1 were upregulated and coexpressed in peripheral CD4+ and CD8+ T cells from BLV-infected cattle. Blockade by anti-bovine TIM-3 monoclonal antibody increased CD69 expression on T cells and gamma interferon (IFN-γ) production from peripheral blood mononuclear cells from BLV-infected cattle. A syncytium formation assay also demonstrated the antiviral effects of TIM-3 blockade against BLV infection. The combined inhibition of TIM-3 and PD-1 pathways significantly enhanced IFN-γ production and antiviral efficacy compared to inhibition alone. In conclusion, the combined blockade of TIM-3 and PD-1 pathways shows strong immune activation and antiviral effects and has potential as a novel therapeutic method for BLV infection. IMPORTANCE Enzootic bovine leukosis caused by bovine leukemia virus (BLV) is an important viral disease in cattle, causing severe economic losses to the cattle industry worldwide. The molecular mechanisms of BLV-host interactions are complex. Previously, it was found that immune checkpoint molecules, such as PD-1, suppress BLV-specific Th1 responses as the disease progresses. To date, most studies have focused only on how PD-1 facilitates escape from host immunity in BLV-infected cattle and the antiviral effects of the PD-1 blockade. In contrast, how T-cell immunoglobulin domain and mucin domain-3 (TIM-3), another immune checkpoint molecule, regulates anti-BLV immune responses is rarely reported. It is also unclear why PD-1 inhibition alone was insufficient to exert anti-BLV effects in previous clinical studies. In this study, the expression profile of TIM-3 in T cells derived from BLV-infected cattle suggested that TIM-3 upregulation is a cause of immunosuppression in infected cattle. Based on these results, anti-TIM-3 antibody was used to experimentally evaluate its function in influencing immunity against BLV. Results indicated that TIM-3 upregulation induced by BLV infection suppressed T-cell activation and antiviral cytokine production. Some T cells coexpressed PD-1 and TIM-3, indicating that simultaneous inhibition of PD-1 and TIM-3 with their respective antibodies synergistically restored antiviral immunity. This study could open new avenues for treating bovine chronic infections.

Primary Results of Stimulus-MDS1: A Randomized, Double-Blind, Placebo-Controlled Phase II Study of TIM-3 Inhibition with Sabatolimab Added to Hypomethylating Agents (HMAs) in Adult Patients with Higher-Risk Myelodysplastic Syndromes (MDS)

Primary Results of Stimulus-MDS1: A Randomized, Double-Blind, Placebo-Controlled Phase II Study of TIM-3 Inhibition with Sabatolimab Added to Hypomethylating Agents (HMAs) in Adult Patients with Higher-Risk Myelodysplastic Syndromes (MDS)

EXTH-58. UNRAVELING THE MECHANISMS UNDERLYING THE THERAPEUTIC EFFICACY OF TIM-3 BLOCKADE IN DIPGS

Abstract Diffuse intrinsic pontine glioma (DIPG) is an aggressive brain tumor and the leading cause of pediatric death caused by cancer. Despite great strides in the understanding of this disease, the survival is still dismal. One of the objectives of our lab is to modulate the tumor microenvironment (TME) towards a proinflammatory phenotype to render these tumors amenable to immunotherapy. TIM-3 is a member of the TIM-family of immunoregulatory proteins expressed on multiple immune cell types, including T-cells, NK, myeloid populations, and microglia, regulating adaptive and innate immunity. In silico assessment of TIM-3 expression in DIPG mRNA and single-cell datasets showed a robust expression of this gene in tumor cells and microglia uncovering this molecule as a potential target in DIPGs. In vivo studies showed that TIM-3 blockade with an antibody significantly increased the overall survival of two DIPG immunocompetent orthotopic models, led to long-term survivors (50%), and showed immune resident memory. TIM-3 inhibition resulted in a significant increase in the number and proliferative state of microglia, NK, and CD8+ cells and higher levels of IFNγ, GrzB and TNFα corresponding to NK and T-cell activate phenotypes. Interestingly, there was a decrease in the Treg population, which causes an increase in the pro-inflammatory CD8/Treg ratio. Chemokine studies demonstrated an augmentation of CCL5, CCL2 chemotactic cytokines, and CXCL10/IL-1β/IFN-γ pro-inflammatory axis in the tumor microenvironment of treated-mice. Additionally, DCs, CD4+, and CD8+ cells were increased in treated draining lymph nodes and of functional significance, expressed higher amounts of pro-inflammatory cytokines than in control mice. Interestingly, the depletion of the different immune populations did not completely abrogate the treatment efficacy indicating a residual although significant TIM-3 effect in the tumor. In conclusion, these data uncover TIM-3 as a potential target for the treatment of DIPG and its potential as an immune regulator of DIPG TME.

Inhibition of Melanoma Cell-Intrinsic Tim-3 Stimulates MAPK-Dependent Tumorigenesis.

Tim-3 is a growth-suppressive receptor intrinsic to melanoma cells, the blockade of which promotes MAPK-dependent tumorigenesis and thus counteracts antitumor activity of T-cell-directed Tim-3 inhibition.

Abstract 615: Tim-3 as an immune therapy target, mechanism and action, and prognostic values with its ligands in patient stratification

Abstract BACKGROUND: Immune checkpoint inhibitors (ICIs) have revolutionized the cancer therapy landscape in recent years. Despite the success of the checkpoint blockade strategies targeting cytotoxic T lymphocyte antigen 4 (CTLA-4) and programmed death receptor 1 (PD-1), many cancer patients cannot benefit from these therapies. T cell immunoglobulin and mucin domain 3 (TIM-3) has emerged as one of the next generation ICI targets, with potentially lower toxicity and higher safety compared to CTLA-4 and PD-1 blockades. TIM-3 is widely expressed in different immune lineages playing various roles such as mediating immune tolerance and regulating innate immune response. However, the mechanism of action of TIM-3 inhibition in different malignancies is not completely understood. Whether the expression and genomic status of TIM-3 and its ligands, Ceacam-1, galectin-9, HMGB1 and phosphatidyl serine (PtdSer) are associated with clinical outcome or any indication would be essential for patient selection for TIM-3 targeting therapies. METHODS: Human PD-1/TIM-3 double-knock-in mice (PD-1/TIM-3 dKI HuGEMM™) engrafted with CT26.WT tumor model were used to test human PD-1 antibody (Keytruda) and TIM-3 antibody (MBG453). Immune phenotyping of blood, tumor draining lymph node (TDLN) and spleen tissues were assessed by flow cytometry at different time points after dosing. Cytokine levels in serum were measured by MSD assays at 48 hours post the 3rd dose and at study termination. Patient genomic and clinical data for various cancer types such as colorectal adenocarcinoma and pancreatic adenocarcinoma were collected for prognostic biomarker analysis. RESULTS: In the in vivo efficacy study of single and combination treatment with Keytruda and MBG453, we observed that NK cells were induced by anti-TIM-3 treatment, alone and in combination, in both blood and TDLN. This indicates TIM-3 blockade may lead to NK cell proliferation in the TME to enhance tumor killing. Transcriptomic analysis on thousands of patients from TCGA showed that high expression of TIM-3 was highly associated with MSI-H and MSI/CIMP subtypes of colorectal adenocarcinoma, suggesting the potential of Tim-3 target therapy in combination with PD-1 blockade in colorectal cancers. By survival analysis, we observe that one of the TIM-3 ligands, HMGB1, expression is associated with patient OS and PFS with pancreatic adenocarcinoma, but not in colorectal cancers. Furthermore, TIM-3 expression was associated with many immune cell signatures, including macrophages, dendritic cells, CD8+ memory T cells, CD4+ memory T cells and Tregs in both colorectal and pancreatic adenocarcinoma. CONCLUSIONS: Evaluation in preclinical model demonstrated that TIM-3 blockade may cause NK cell proliferation to enhance anti-tumor immunity. In addition, the expression and genomic alteration of TIM-3 and its ligand have prognostic values for certain cancers. Citation Format: Jia Xue, Yu Zhang, Xianfei He, Henry Q. Li, Sheng Guo. Tim-3 as an immune therapy target, mechanism and action, and prognostic values with its ligands in patient stratification [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 615.

The Potential of T Cell Immunoglobulin and Mucin-Domain Containing-3 (Tim-3) in Designing Novel Immunotherapy for Bladder Cancer.

Targeting inhibitory receptors on T cells in the tumor sites can promote effective anti-tumor immunity in bladder cancer. Unfortunately, the main dilemma is that a large number of patients remain refractory to CTLA-4, PD-1, and PD-L1 blockade therapies. T-cell immunoglobulin and mucin domain 3 (Tim-3) is an inhibitory receptor expressed on T cells and innate immune cells. Both in vivo and in vitro data from patients with advanced cancers support the role of Tim-3 inhibition in satisfactory anti-tumor immunity. In bladder cancer, the expression level of Tim-3 significantly increases with advanced pathological grade and T stage. Therefore, rationality implies that designing novel monoclonal antibodies reactive with Tim-3 alone or in combination with other checkpoint inhibitors may indicate a favorable response in bladder cancer. Here, we aimed to investigate the possibility of targeting Tim-3 as a novel anti-cancer treatment for bladder cancer.

Genetic Mutations of Tim-3 Ligand and Exhausted Tim-3+ CD8+ T Cells and Survival in Diffuse Large B Cell Lymphoma.

Tim-3 is a promising target for antitumor immunotherapy. A number of clinical trials are evaluating the efficacy of anti-Tim-3 therapies as a single agent or combinations in solid tumors and haematologic malignancies. However, there remains a considerable lack of data on Tim-3 signalling, especially the genetic characteristics and immune microenvironment, in diffuse large B cell lymphoma (DLBCL). Herein, we identified three genetic mutations in galectin-9, a major ligand of Tim-3, in six patients with DLBCL (6/188, 3.2%) that were not detected in the COSMIC database. The Oncomine database showed that the mRNA levels of Tim-3 were higher in DLBCL cells than those in normal B cells. Multiplexed immunofluorescence revealed that patients with Tim-3-expressing tumor-infiltrating lymphocytes (Tim-3+ TILs) exhibited poor outcomes than those with Tim-3− TILs (p = 0.041). The median survival times of these patients were 65.0 (95% confidence interval (CI): 71.2–88.6) and 79.9 months (95% CI: 54.4–75.6), respectively. Furthermore, we defined a novel subtype of exhausted T cells, named as exhausted Tim-3+ CD8+ T cells, and found that patients with exhausted Tim-3+ CD8+ T cells (median survival, 62.8 months, 95% CI: 50.0–75.6) exhibited shorter survival than those with nonexhausted Tim-3− CD8+ T cells (median survival, 82.5 months, 95% CI: 72.0–92.9; p = 0.034). Overall, these findings provide the genetic status of the Tim-3 ligand in DLBCL. Patients with Tim-3+ TILs and exhausted Tim-3+ CD8+ T cells exhibited inferior survival, thus highlighting the possibility of potential therapeutic applications of the inhibition of Tim-3 alone or in combination with other immune checkpoints for treatment of patients with DLBCL.

Abstract 6565: Impact of TIM-3 modulation on the sensitivity of anti-PD-1

Abstract Concurrent inhibition of TIM-3 and PD-1 may have enhanced anti-tumor effects. In this study, computational models were established to identify factors involved in the interactions between these two immunotherapeutic targets. Abstracts published on TIM-3, PD-1, PD-L1, anti-PD-1/PD-L1 were downloaded from PubMed, and analyzed by natural language processing and text mining. Interactions among genes, compound/therapies, cell/animal models, pathway/disease, and clinical studies were retrieved from the text. Networks were constructed to simulate effects of TIM-3 inhibition on anti-PD-1. The TIM-3/anti-PD-1 network consists of 57 nodes, 175 interactions with 11,626 possible regulatory paths from TIM-3 to anti-PD-1. Overall, 6142 paths are associated with the agonistic interactions between TIM-3 inhibition and anti-PD-1, while 5484 are antagonistic. Factors associated with the enhancement of anti-PD-1 by TIM-3 inhibition include up-regulation of IL-2, IFN-gamma, Th1 cells/cytokines, CD8+ T cells or NK cells, and down-regulation of IL-10, NFkB-p65, COX2 or EMT. Treatments with either EGFR inhibitors (e.g. gefitinib), anti-CTLA-4, IL-2 agonist or radiation therapy may enhance the combination efficacy of TIM-3 and PD-1 inhibitors. On the other hand, CTLA-4, galectin-9, Wnt/beta-catenin or Tregs combined with TIM-3 over-expression may confer resistance to anti-PD-1. PD-L1 is a validated biomarker for anti-PD-1 response and its expression can be correlated with TIM-3. Similar text-mining approach was adopted to construct the TIM-3/PD-L1 network which consists of 65 nodes, 165 interactions and 1372 paths from TIM-3 to PD-L1. Up-regulation of either galectin-9, WNT/beta-catenin or Tregs by TIM-3 results in the induction of PD-L1. As the network analyses revealed, interactions between TIM-3 and PD-L1 are context dependent, and assessment of the effect of TIM-3 inhibition on anti-PD-1 requires profiling of multiple factors along the regulatory and signaling pathways. The TIM-3/PD-L1 and TIM-3/anti-PD-1 networks prove to be useful tools to facilitate the therapeutic development of TIM-3 and PD-1 inhibitors, especially for selecting patients with potential agonistic effects of this promising drug combination. Citation Format: Jian Zhu, Rebecca L. Zhu. Impact of TIM-3 modulation on the sensitivity of anti-PD-1 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6565.

TIM3 comes of age as an inhibitory receptor.

T cell immunoglobulin and mucin domain-containing protein 3 (TIM3), a member of the TIM family, was originally identified as a receptor expressed on interferon-γ-producing CD4+ and CD8+ T cells. Initial data indicated that TIM3 functioned as a 'co-inhibitory' or 'checkpoint' receptor, but due to the lack of a definable inhibitory signalling motif, it was also suggested that TIM3 might act as a co-stimulatory receptor. Recent studies have shown that TIM3 is part of a module that contains multiple co-inhibitory receptors (checkpoint receptors), which are co-expressed and co-regulated on dysfunctional or 'exhausted' T cells in chronic viral infections and cancer. Furthermore, co-blockade of TIM3 and programmed cell death 1 (PD1) can result in tumour regression in preclinical models and can improve anticancer T cell responses in patients with advanced cancers. Here, we highlight the developments in understanding TIM3 biology, including novel ligand identification and the discovery of loss-of-function mutations associated with human disease. In addition, we summarize emerging data from human clinical trials showing that TIM3 indeed acts as a 'checkpoint' receptor and that inhibition of TIM3 enhances the antitumour effect of PD1 blockade.

1202P - Phase (Ph) II study of MBG453 + spartalizumab in patients (pts) with non-small cell lung cancer (NSCLC) and melanoma pretreated with anti–PD-1/L1 therapy

1202P - Phase (Ph) II study of MBG453 + spartalizumab in patients (pts) with non-small cell lung cancer (NSCLC) and melanoma pretreated with anti–PD-1/L1 therapy

Abstract 673: Impact of PD-L1 feedback modulation on the sensitivity of anti-PD-1/PD-L1

Abstract The long-term benefits of anti-PD-1/PD-L1 are limited in most cancer patients. Resistance and insensitivity are major challenges of this therapeutic class. Feedback regulation of PD-L1 could serve as a mechanism of drug resistant, especially negative feedback loops where inhibition of PD-L1 may lead to up-regulation of its own expression and function. In this study, computational models were established to investigate PD-L1 feedback modulations to facilitate discovery of biomarkers for drug sensitivity and to inform design of drug combination studies. As of October 2018, over 10,000 publications on cancer and immunosuppressive factors (e.g. PD-1, PD-L1, CTLA4, IDO1, TIM-3, LAG3, TIGIT) were found in PubMed. About 2500 abstracts of these articles were downloaded and systematically analyzed through natural language processing and text mining. Information on gene/drug, pathway/disease along with results from cell/animal/clinical studies were extracted, standardized and integrated into a directed graph-based database. Algorithms were designed to construct networks and identify PD-L1 feedback loops (FBLs). Fisher's Exact tests were performed to evaluate the significance of factors involved in (+) and (-)FBLs. The computationally generated PD-L1 feedback network comprises 102 nodes, 216 interactions with 1080 possible FBLs (502 positive vs 578 negative). In particular, up-regulation of IL10, pEGFR, SRC, HIF1A, ISX, NF-kappaB, pSMAD2/SMAD2, IFN-gamma were strongly associated with up-regulation of PD-L1 upon inhibition of PD-1/PD-L1 (p<0.0001, (-)FBLs). Interestingly, TIM-3, a potential IO target and commonly up-regulated by anti-PD-1/PD-L1, is involved in both (+) and (-)FBLs. Inhibition of TIM-3 (e.g. anti-TIM-3) could decrease IL-10 in Tregs and enhance efficacy of anti-PD-1/PD-L1, but meanwhile, anti-TIM-3 could activate NF-kappaB, IRF7 and STAT1, leading to up-regulation of PD-L1 expression and potential resistance to anti-PD-1/PD-L1. In conclusion, the literature-based network analysis proved to be effective to identify potential regulatory pathways driving resistance to immunotherapies, to prioritize the selection of biomarkers, and to assess risk of drug combinations. Particularly, through the analysis of feedback regulations of PD-L1, novel therapeutic approaches to boost efficacy of anti-PD-1/PD-L1 could be postulated. Targeting genes involved in both (+) and (-)FBLs of PD-L1, such as TIM-3, needs to be carefully evaluated. Citation Format: Jian Zhu, Rebecca L. Zhu. Impact of PD-L1 feedback modulation on the sensitivity of anti-PD-1/PD-L1 [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 673.

A Deficit of CEACAM-1-Expressing T Lymphocytes Supports Inflammation in Primary Progressive Multiple Sclerosis.

The immune regulatory mechanisms that modulate Th1 and Th17 immune responses are altered in multiple sclerosis (MS). The inhibitory TIM-3/Gal-9 pathway, in particular, is impaired in primary progressive MS (PPMS). Recent results showed that carcinoembryonic Ag-related cell adhesion molecule 1 (CEACAM-1), a molecule expressed on activated T lymphocytes, endows TIM-3 with inhibitory function and facilitates the maturation and cell surface expression of TIM-3. We analyzed by flow cytometry CEACAM-1 expression on myelin basic protein (MBP)-stimulated CD4+ and CD8+ T lymphocytes of 56 MS patients with a diagnosis of either PPMS (n = 16), relapsing-remitting MS (n = 20), or benign MS (n = 20) and 40 age- and sex-matched healthy controls. The expression of TIM-3 and annexin V (AV) as well as the production of IFN-γ and the intracellular concentration of HLA-B-associated transcript 3 (Bat3), a molecular adaptor that binds the intracellular tail of TIM-3 promoting both proliferation and proinflammatory cytokine production, were analyzed as well in the same cells. Results showed the following in PPMS: 1) CD4+/CEACAM-1+, CD4+/TIM-3+, CD8+/TIM-3+, CD4+/CEACAM-1+/TIM-3+, and CD8+/CEACAM-1+/TIM-3+ T lymphocytes as well as CEACAM-1 mean fluorescence intensity on CD4+ T lymphocytes were significantly reduced; 2) apoptotic CD4+/AV+/CEACAM-1+ and CD8+/AV+/CEACAM-1+ T lymphocytes were significantly reduced; and 3) Bat3-expressing CD4+ and CD8+ T cells were significantly increased. Notably, a specular immunologic scenario was seen in benign MS. CEACAM-1 expression is reduced in PPMS; this exacerbates MBP-specific inflammatory T cell response and reduces the apoptosis of MBP-specific T lymphocytes, possibly as a consequence of the upregulation of Bat3 seen in these patients.

Critical role of Tim-3 mediated autophagy in chronic stress induced immunosuppression

BackgroundPsychological and physical stress can either enhance or suppress immune functions depending on a variety of factors such as duration and severity of stressful situation. Chronic stress exerts a significantly suppressive effect on immune functions. However, the mechanisms responsible for this phenomenon remain to be elucidated. Autophagy plays an essential role in modulating cellular homeostasis and immune responses. However, it is not known yet whether autophagy contributes to chronic stress-induced immunosuppression. T cell immunoglobulin and mucin domain 3 (Tim-3) has shown immune-suppressive effects and obviously positive regulation on cell apoptosis. Tim-3 combines with Tim-3 ligand galectin-9 to modulate apoptosis. However, its impact on autophagy and chronic stress-induced immunosuppression is not yet identified.ResultsWe found remarkably higher autophagy level in the spleens of mice that were subjected to chronic restraint stress compared with the control group. We also found that inhibition of autophagy by the autophagy inhibitor 3-methyladenine (3-MA) significantly attenuated chronic stress-induced alterations of pro-inflammatory and anti-inflammatory cytokine levels. We further elucidated that 3-MA dramatically inhibited the reduction of lymphocyte numbers. Moreover, chronic stress dramatically enhanced the expression of Tim-3 and galectin-9. Inhibition of Tim-3 by small interfering RNA against Tim-3 significantly decreased the level of autophagy and immune suppression in isolated primary splenocytes from stressed mice. In addition, α-lactose, a blocker for the interaction of Tim-3 and galectin-9, also decreased the autophagy level and immune suppression.ConclusionChronic stress induces autophagy, resulting with suppression of immune system. Tim-3 and galectin-9 play a crucial regulatory role in chronic stress-induced autophagy. These studies suggest that Tim-3 mediated autophagy may offer a novel therapeutic strategy against the deleterious effects of chronic stress on the immune system.

Tumor cell-intrinsic Tim-3 promotes liver cancer via NF-κB/IL-6/STAT3 axis.

T-cell immunoglobulin and mucin-domain containing-3 (Tim-3), mediating immune exhaustion in tumor microenvironment, has become a promising target for tumor therapy. However, the exact mechanisms for tumor cell-intrinsic Tim-3 in tumor development and its potential contribution in Tim-3-targeted therapy strategy have not been elucidated yet. In this study, we showed that human liver cancer tissues contained high ratio of Tim-3-expressing hepatocytes, and cytokines rich in tumor microenvironment and HBV involved in Tim-3 upregulation in malignant hepatocytes. We demonstrated that hepatocyte-specific Tim-3 overexpression enhances tumor cell growth, whereas Tim-3 inhibition on malignant hepatocytes by anti-Tim-3 antibodies or RNAi suppresses tumor growth both in vitro and in Tim-3 knockout mice. Mechanistically, the hepatocyte-Tim-3 receptor activates NF-κB phosphorylation, which in turn stimulates IL-6 secretion and STAT3 phosphorylation. Our results identify tumor cell-intrinsic functions of Tim-3 in tumorigenesis and suggest that blocking Tim-3 in tumor cells might contribute to the clinical efficacy of anti-Tim-3 antibody treatment in the future tumor therapy.

Abstract 2932: Immuno-inhibitory pathway, TIM-3/Galectin-9, in lung adenocarcinoma: clinical and in vitro analysis

Abstract Introduction and Purpose: Recently, immune checkpoint therapy has been incorporated into treatment of solid cancers including lung cancer, resulted in prolonged survival benefit. Checkpoint inhibitors target on PD-1/PD-L1 or CTLA-4 immuno-inhibitory molecules in immune or cancer cells. However, checkpoint therapy in lung adenocarcinoma (LAD) showed less than 20% response rate, suggesting existence of other immuno-inhibitory pathways. Therefore, we focused on T cell immunoglobulin and mucin domain 3 (TIM-3) on the CD4 and CD8 T-cell surface and its ligand Galectin-9 in tumor cells, and analyzed the expression and function in LAD cells and tissues. Materials and Methods: TILs and PBMCs were obtained from 11 patients with LAD, and 194 LAD tissues resected surgically were analyzed. Expression of immuno-inhibitory molecules (PD-1, TIM-3, BTLA, LAG-3) was examined by flow cytometry using FACS on TILs and PBMCs, and PD-L1 and Galectin-9 expressions in LAD tissues was analyzed by immunohistochemistry using tissue microarray. To investigate soluble Galectin-9 released from LAD cells, EGFR-mutated PC-9 and EGFR-wild-type OU-LC-SK cells were treated with EGFR-TKI afatinib at a concentration of 10 nM for 3 days. Then, the amount of Galectin-9 in culture supernatant was measured by ELISA. To investigate T-cell apoptosis induction by Galectin-9, established XAGE1-specifiic CD8 cloned T-cells were incubated with Galectin-9 protein for 8 hrs. Results: Increased expression in TILs compared to PBMCs was observed on PD-1 and TIM-3, but not on BTLA or LAG-3 in CD4 and CD8 T-cells. In LAD tissues, the frequencies of high PD-L1 and Galectin-9 expression in the tumor cell membrane or cytoplasm were 49% and 31%, respectively (in squamous cell carcinoma, the frequencies were 32% and 16%, respectively). Furthermore, correlated expression of PD-L1, Galectin-9 and CD3 (T-cell infiltration) was observed at the periphery of the tumor nest. Those findings suggest the relevance of the PD-1/PD-L1 and TIM-3/Galectin-9 pathways in the tumor microenvironment of LAD. Next, soluble Galectin-9 released from the LAD cells was measured. Galectin-9 was detected only in the medium of EGFR-mutated LAD cells following treatment with afatinib. Moreover, apoptosis was induced in TIM-3-positive CD8 T-cell clones following interaction with Galectin-9 protein and this was inhibited by the addition of anti-Galectin-9 or an anti-TIM-3 antibody. The findings suggested that a significant amount of Galectin-9 could be released from LAD cells and induced T-cell apoptosis in tumor microenvironment. Conclusions: Our results suggested the relevance of the PD-1/PD-L1 and TIM-3/Galectin-9 immuno-inhibitory pathways in the tumor microenvironment of LAD, and that release of soluble Galectin-9 from LAD cells could negatively regulate T-cell function. For successful immune checkpoint therapy in LAD, simultaneous inhibition of TIM-3/Galectin-9 pathway may be needed. Citation Format: Mikio Oka, Yoshihiro Ohue, Koji Kurose, Yumi Nishio, Midori Isobe, Eiichi Nakayama. Immuno-inhibitory pathway, TIM-3/Galectin-9, in lung adenocarcinoma: clinical and in vitro analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2932. doi:10.1158/1538-7445.AM2017-2932