T-cell Immunoglobulin And Mucin-domain Containing-3 Research Articles

Abstract 933: Pre-clinical characterization of TIM-3 antibody NB002

Abstract Targeting immune checkpoint molecules PD1 and CTLA-4 by activating the immune system have improved the lives of many cancer patients. However, this type of immunotherapy is not effective for all patients, suggesting that there are additional mechanisms of immune evasion. T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) has been reported to be expressed on exhausted T and NK cells within the tumor microenvironment. The data in many preclinical models suggest that targeting TIM-3 pathway might provide an additional immune modulation of this anti-tumor axis in cancer patients. We have developed a novel monoclonal antibody NB002 against TIM-3,which forms a high-affinity interaction with a unique epitope. NB002 can enhance IFN-gamma secretion and proliferation of these exhausted T cells that has increased TIM-3 expression. Moreover, TIM-3 is constitutively expressed on NK cells. TIM-3 blockade could enhance in vitro cytotoxic activity of NK cells. Altogether, our data shows that NB002 is capable of targeting exhausted T cells and NK cells resulting activation of antitumor immunity. Further preclinical assessment in vivo and in vitro for solid tumors will support clinical development of NB002. Citation Format: Xin Dong, Jannie Dong, Jun Ma, Lianqi Zhao, Haojie Wang, Binbin Wang, Qian Gao, Hui Pan, Dong Wang, Liegang Shao. Pre-clinical characterization of TIM-3 antibody NB002 [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 933.

Immune Checkpoint Expression on Immune Cells of HNSCC Patients and Modulation by Chemo- and Immunotherapy.

Endogenous control mechanisms, including immune checkpoints and immunosuppressive cells, are exploited in the process of tumorigenesis to weaken the anti-tumor immune response. Cancer treatment by chemotherapy or immune checkpoint inhibition can lead to changes of checkpoint expression, which influences therapy success. Peripheral blood lymphocytes (PBL) and tumor-infiltrating lymphocytes (TIL) were isolated from head and neck squamous cell carcinoma (HNSCC) patients (n = 23) and compared to healthy donors (n = 23). Immune checkpoint expression (programmed cell death ligand 1 (PD-1), tumor necrosis factor receptor (TNFR)-related (GITR), CD137, tumor necrosis factor receptor superfamily member 4 (TNFRSF4) (OX40), t-cell immunoglobulin and mucin-domain containing-3 (TIM3), B- and T-lymphocyte attenuator (BTLA), lymphocyte-activation gene 3 (LAG3)) was determined on immune cells by flow cytometry. PD-L1 expression was detected on tumor tissue by immunohistochemistry. Immune cells were treated with immuno- and chemotherapeutics to investigate treatment-specific change in immune checkpoint expression, in vitro. Specific changes of immune checkpoint expression were identified on PBL and TIL of HNSCC patients compared to healthy donors. Various chemotherapeutics acted differently on the expression of immune checkpoints. Changes of checkpoint expression were significantly less pronounced on regulatory T cells compared to other lymphocyte populations. Nivolumab treatment significantly reduced the receptor PD-1 on all analyzed T cell populations, in vitro. The specific immune checkpoint expression patterns in HNSCC patients and the investigated effects of immunomodulatory agents may improve the development and efficacy of targeted immunotherapy.

Bgb-A425, an investigational anti-TIM-3 monoclonal antibody, in combination with tislelizumab, an anti-PD-1 monoclonal antibody, in patients with advanced solid tumors: A phase I/II trial in progress.

TPS3146 Background: While immune surveillance plays a critical role in preventing tumor proliferation and metastasis, tumors develop resistance mechanisms to suppress and/or escape the immune system. T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) and programmed cell death protein-1 (PD-1) function as immune checkpoint receptors on tumor-infiltrating lymphocytes. Overlap in expression and function suggests TIM-3 and PD-1 cooperate to maximize effector T-cell exhaustion, leading to a decreased antitumor immune response. Although blockade of TIM-3 alone is unlikely to result in an efficacious antitumor immune response, combined TIM-3/PD-1 blockade may enhance the antitumor properties of anti-PD-1 therapies alone. BGB-A425 is an investigational IgG1-variant monoclonal antibody against TIM-3. Tislelizumab, an anti-PD-1 antibody, was engineered to minimize binding to FcɣR on macrophages in order to abrogate antibody-dependent phagocytosis, a mechanism of T-cell clearance and potential resistance to anti-PD-1 therapy. This phase 1/2 study will assess the safety/tolerability, pharmacokinetic (PK) profile, and antitumor activity of BGB-A425 in combination with tislelizumab in patients with advanced solid tumors. Methods: This is an open-label phase 1/2 study (NCT03744468) of BGB-A425 in combination with tislelizumab in patients with histologically/cytologically confirmed advanced, metastatic, unresectable solid tumors. Phase 1 will determine the recommended phase 2 dose (RP2D) for combination treatment; phase 2 will assess the antitumor effects of the combination in select tumor types. In phase 1, up to 42 patients will be enrolled into sequential cohorts of increasing doses of intravenous (IV) BGB-A425 in combination with tislelizumab 200 mg IV, based on a 3+3 study design. During Cycle 1, patients will receive BGB-A425 alone on Day 1 followed by tislelizumab alone on Day 8. If no dose-limiting toxicities are observed, patients will receive both BGB-A425 and tislelizumab sequentially on Day 29 and every 21 days thereafter. Once the RP2D is determined, the combination therapy will be evaluated in up to 120 patients with select tumor types in phase 2. Safety/tolerability profile and RP2D determination (phase 1) and objective response rate per RECIST v1.1 (phase 2) are primary objectives; secondary objectives include antitumor activity, PK profile, and immunogenicity of combination therapy. Clinical trial information: NCT03744468 .

Dual but not single PD-1 or TIM-3 blockade enhances oncolytic virotherapy in refractory lung cancer

BackgroundProgrammed cell death 1 (PD-1)/programmed death ligand 1 (PD-L1) blockade therapy fails in the majority of patients with cancer. Oncolytic viruses represent a new class of therapeutic agents, yet the...

Differential expression of TIM-3 in circulation and tumor microenvironment of colorectal cancer patients

T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) is an inhibitory immune checkpoint, which suppresses anti-tumor immune responses. TIM-3 expression on different immune cells in periphery and tumor microenvironment (TME) of colorectal cancer (CRC) patients has not been fully investigated. We found that TIM-3 was mainly expressed on monocytic myeloid cells (MMCs) and antigen-presenting cells (APCs) in circulation but was mainly expressed on T cells and APCs in the TME. Additionally, TIM-3− T cells co-expressed higher levels of PD-1 than TIM-3+ T cells in normal tissue. In contrast, TIM-3+ T cells in the TME showed significantly higher PD-1 expression. Interestingly, there was a trend towards increased levels of TIM-3+ APCs with disease stages; however, levels of TIM-3+ T cells were decreased with disease stages in the TME. This study shows the differential expression of TIM-3 on different immune cells in circulation and TME of CRC patients, and their associations with disease stages.

Significance of TIM-3 Expression in Resected Esophageal Squamous Cell Carcinoma

T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) is a promising checkpoint. However, its features and prognostic value remain undetermined in esophageal squamous cell carcinoma (ESCC). This study evaluated the prognostic value of TIM-3 expression and its relationship with programmed cell death 1 (PD-1) and CD8+ tumor-infiltrating lymphocytes (TILs) in patients with surgically resected ESCC. Expression levels of TIM-3, PD-1, and CD8+ TILs in ESCC were determined by immunohistochemistry. The association between clinicopathologic features or clinical outcomes and TIM-3 expression was analyzed. A total of 183 patients with ESCC who had undergone esophagectomy without implementation of neoadjuvant therapy at the Second Affiliated Hospital of Soochow University in Suzhou, China from January 2009 to December 2014 were included. PD-1 positivity (P=.032) and high CD8+ TIL density (P= .035) significantly correlated with positive TIM-3 expression. TIM-3 positivity was an independent risk factor for recurrence-free survival (RFS) (P < .001) and overall survival (OS) (P < .001). Subgroup analysis revealed that the TIM-3+PD-1+CD8 low group had the worst RFS and OS, whereas the TIM-3-PD-1-CD8 high group had the best RFS and OS (RFS: log-rank test P < .001; OS: log-rank test P < .001). Positive TIM-3 expression was associated with PD-1 positivity and high CD8+ TIL density and was an independent risk factor for RFS and OS in ESCC. Furthermore, the combination of TIM-3 and PD-1 expression or CD8+ TIL density could further stratify patients into different groups with distinct prognosis.

Tumor-infiltrating podoplanin+ cells in gastric cancer: clinical outcomes and association with immune contexture

ABSTRACT Podoplanin (PDPN) has been proved to have significant immunoregulatory effects in several types of malignancies and is considered to be a novel immune checkpoint molecule. However, the clinical significance of PDPN and its potential influence on immune contexture in gastric cancer remain obscure. Here, we aimed to investigate the clinical outcomes and immunoregulatory role of tumor-infiltrating PDPN+ cells (tPDPNs) in gastric cancer. A total of 454 tumor tissue microarray specimens and 68 fresh tumor tissues of gastric cancer patients from Zhongshan Hospital, and transcriptional data of 293 gastric cancer patients from The Cancer Genome Atlas were included. We demonstrated that tPDPNs high subgroup experienced worse overall survival and disease-free survival, and indicated inferior therapeutic responsiveness to fluorouracil-based adjuvant chemotherapy (ACT) in gastric cancer. The abundance of tPDPNs was correlated with an immunoevasive contexture characterized by pro-tumor macrophage and dysfunctional CD8+ T cell infiltration. Moreover, dysfunctional CD8+ T cells in tPDPNs high subgroup exhibited decreased interferon-γ, granzyme B and perforin-1 expression yet elevated programmed cell death-1 (PD-1) and T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) expression. Stratification of gastric cancer patients into different risk groups based on tPDPNs and CD8+ T cells showed distinct prognosis, responsiveness to ACT and molecular characteristics. This study revealed that the abundance of tPDPNs could identify an immunoevasive contexture and might be applied as an independent predictor for poor prognosis and suboptimal ACT responsiveness. Thus, we recommended tPDPNs as a promising therapeutic target in gastric cancer.

PD-L1, PD-1, LAG-3, and TIM-3 in Melanoma: Expression in Brain Metastases Compared to Corresponding Extracranial Tumors.

BackgroundMetastatic melanoma to the brain carries a particularly poor prognosis that may be associated with an attenuated antitumor response in the presence of central nervous system malignancies. Thus, the development of brain metastases could theoretically accelerate cancer progression both locally and systemically. Although dysregulation of checkpoint markers, such as programmed death-ligand 1 (PD-L1), programmed cell death receptor 1 (PD-1), lymphocyte activation gene 3 (LAG-3), and T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), have been implicated in immune dysfunction, the exact relationship between these markers and brain tumor-mediated immune suppression remains unclear. Thus, the objective of this study was to explore whether there exists a differential expression of the above checkpoint markers in the intracranial milieu as compared to tumors in the periphery, which may shed light on the mechanism behind the diminished antitumor response.MethodsWe identified nine patients with extracranial melanomas and matched intracranial metastases. Formalin-fixed, paraffin-embedded slides were stained for PD-L1, PD-1, LAG-3, and TIM-3 via immunohistochemistry. Qualitative analysis was performed to assess the staining of the markers in the neoplastic and lymphocytic cells, which were the two cell lineages in each biopsy. ResultsExpression of PD-1 and TIM-3 between extracranial and intracranial tumoral sites was conserved. Specifically, in lymphocytes, PD-1 expression was observed in 100% of extracranial and 100% of intracranial slides, whereas TIM-3 expression was seen in 33.33% of extracranial and 33.33% of intracranial slides. Neither marker stained tumor cells, as expected. PD-L1 showed a slight variation in staining between sites, with lymphocyte staining in 100% of extracranial and 88.89% of intracranial slides, and the same percentages per site for tumor cells. The greatest variability was observed in LAG-3 lymphocyte staining, with staining in 77.78% of extracranial and 33.33% of intracranial slides. No LAG-3 staining of tumor cells was noted, as expected.ConclusionPreliminary analysis revealed the conservation of PD-L1, PD-1, LAG-3, and TIM-3 expression intra- and extracranially. This could suggest that these markers are important in maintaining an immunosuppressive phenotype at both sites. Another possibility is that this pattern of expression is associated with patients who develop brain metastasis, as this was the only subset of patients included in this study. Interestingly, LAG-3 staining of lymphocytes appeared more prominent in extracranial over intracranial tumors. Future studies should include more samples to draw out potential patterns masked by the small sample size, as well as to compare checkpoint expression in other patient groups, such as those with non-brain metastasis or those with no metastasis at all.

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.

TIM-3 expression and its association with overall survival in primary osteosarcoma.

T-cell immunoglobulin and mucin domain-containing-3 (TIM-3) performs a critical function in immune tolerance by suppressing the activation and proliferation of T cells. TIM-3 serves an important role in tumor progression in a number of carcinomas, including non-small cell lung cancer, hepatitis B virus-associated hepatocellular carcinoma, Langerhans cell sarcoma, head and neck cancer and follicular B cell non-Hodgkin lymphoma. The aim of the present study was to evaluate the possible association of TIM-3 with the prognosis of osteosarcoma. TIM-3 expression was assessed by immunohistochemical analysis in osteosarcoma tissues. The association between TIM-3 expression and prognosis was examined. To assess the association between TIM-3 expression levels and clinicopathological features, a Fisher's exact test was used. TIM-3 overexpression was indicated to be associated with surgical treatment and survival. Kaplan-Meier analysis indicated that TIM-3 is an independent predictor of overall survival, and its overexpression was indicated to be associated with poor prognosis in patients with osteosarcoma. Additionally, reverse transcription-quantitative polymerase chain reaction and western blot analysis were carried out to evaluate TIM-3 expression levels in fresh tumor tissue samples, adjacent-tissue samples, osteosarcoma cell lines, and in an osteoblastic cell line. TIM-3 was indicated to be overexpressed in fresh osteosarcoma tissue samples and in osteosarcoma cell lines. In conclusion, TIM-3 overexpression is associated with poor survival among patients with osteosarcoma and may be a possible therapeutic target in these types of tumors.

Abstract P4-06-10: Immune related gene expression to explore immue escape in primary to metastatic breast cancer transition

Abstract Background: Targeting programmed cell death receptor l pathway (anti-PD-1/PD-L1) demonstrated objective clinical response rates in a subgroup of patients with metastatic breast cancer with overall response rates of up to 20%. However, the vast majority of breast cancer patients is not responding to immune checkpoint blockade. With regard to the diverse tumor biology of breast cancer and recent data, immune evasion is likely mediated by various, coincident mechanisms. Consequently, our objective was to identify patterns of immune responses during the transition of primary to metastatic breast cancer. Methods: We performed comprehensive immunohistochemistry on both tumor and immune cells in two independent, matched cohorts of 67 primary and metastatic breast cancer patients. Further, the analysis was integrated with mRNA expression levels (RT-qPCR) of key regulatory and effector function immune genes and clinical as well as histopathologic parameters. Results: Both, immunohistochemistry and mRNA gene expression profiling of immune genes revealed immunological ignorance, defined as an immunological “cold” tumor by particularly low infiltration of CD8+ T cells and corresponding immune gene and effector function, as a key feature of primary to metastatic breast cancer transition. However, CD8+ T cell infiltration and effector function (Granzyme B, Interferon-γ) where found to be an independent prognostic marker for PFS and OS. Unsupervised hierarchical clustering and regression analysis identified distinct immune response types according to T-cell infiltration, interferon-γ signalling (IFNG), immunosuppression (IDO, TGF-β, FOXP3) and humoral immune responses (Immunoglobulin κ constant; IGKC). Expression of programmed death receptor 1 ligand (PD-L1), and T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) where linked to CD8+ T cells and associated with a better prognosis only in triple negative breast cancer patients. Importantly, induction of CD4+ and CD8+ T cell infiltration as well as humoral immune responses (IGKC, CXCL13) in matched pairs during progression from primary to metastatic disease, where strongly associated with improved distant disease free and overall survival. Conclusion: Our findings demonstrate a switch to a less immunoreactive environment in metastatic compared to matched, primary samples of breast cancer patients. Nevertheless, induction of T-cell mediated immunity during tumor evolution, observed across all biologic subtypes, warrants further research with the overall goal to convert immunologically ignorant breast cancers and to stratify future immunotherapies. Citation Format: Fremd C, Halama N, Wirtz R, Zoernig I, Sinn H-P, Varga Z, Feisst M, Deutsch T, Schuetz F, Schneeweiss A, Jaeger D, Wallwiener M. Immune related gene expression to explore immue escape in primary to metastatic breast cancer transition [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-06-10.

Immune Checkpoints as the Immune System Regulators and Potential Biomarkers in HIV-1 Infection.

Immune checkpoints are several co-stimulatory and inhibitory pathways that regulate T cell immune responses. Most of the discoveries about immune checkpoints were made in cancer research where inhibitory immune checkpoints cause immune exhaustion and down-regulate anti-tumor responses. In addition to cancer, immune checkpoints are exploited in chronic infectious diseases. In human immunodeficiency virus (HIV) infection, the immune checkpoint molecule called programmed cell death protein 1 (PD-1) has been determined as being a major regulatory factor for T cell exhaustion. Recent studies with antibodies blocking either PD-1 ligand 1 (PD-L1) or PD-1 show not only promising results in the enhancement of HIV-specific immune responses but even in reducing the latent HIV reservoir. Apart from the therapeutic target for a functional cure of HIV-1, immune checkpoint molecules might be used as biomarkers for monitoring disease progression and therapeutic response. In this review, we will summarize and discuss the inhibitory immune checkpoint molecules PD-1, cytotoxic T-lymphocyte-associated protein 4 (CTLA4), lymphocyte-activation gene 3 (LAG3), and T cell immunoglobulin and mucin-domain-containing-3 (TIM3) as well as the co-stimulatory molecules CD40L and CD70, including their role in immunity, with a particular focus on HIV infection, and being potential targets for a functional HIV cure.

PO-400 Dual targeting of TIM3 and adenosine produces synergistic improvement in anti-tumour immunity

IntroductionCD8 +Cytotoxic T Lymphocytes (CTL) are able to kill tumour cells. However, their killing is frequently suppressed within the tumour microenvironment, due to the interaction of immune checkpoint receptors on tumour infiltrating CTL with tumour-derived ligands. Drugs which block checkpoints have shown great promise in controlling tumour growth. Nevertheless, there is a high rate of immune-related adverse effects associated with these drugs, thus limiting their use. Moreover, in response to blockade of one receptor, tumours may escape treatment by upregulating other checkpoint pathways. As such, there is a need to identify novel tumour-specific checkpoints, to mitigate autoimmune side-effects; and to target these in combination, to prevent tumour immune evasion.Material and methodsBALB/c mice bearing subcutaneous murine renal carcinomas, expressing Haemagglutinin (HA) from Influenza A/PR/8 as a model tumour antigen were treated as follows: a) Adoptive Transfer of activated TcR transgenic, HA-specific CD8 +T cells (CL4 cells) b) A2a Adenosine-receptor antagonist 200 ug q2d (ZM241385, Santa Cruz) c) Anti-TIM3 mAb (Clone RMT-3, BioXcell), 100 ug q2d. Vehicle and isotype controls were used. Tumour growth was measured and the volume calculated, Volume=0.5 (Length x (Width2)). Flow cytometric analysis of checkpoint expression was carried out using a BD Fortessa and FACS DIVA software. Data was analysed using FlowJo (Treestar). Principal component analysis and ANCOVA were performed using SPSS and R-Studio.Results and discussionsIn our model, although blockade of the A2aR Adenosine receptor produces partial control of tumour growth, tumours do not completely regress. Principal component analysis was used to evaluate expression of other immune checkpoints across 43 treated and untreated tumours. Analyses showed that expression of T-cell immunoglobulin and mucin-domain containing-3 (TIM3) contributed strongly to the variation between treated and control groups. These data suggest that upregulation of TIM3 represents a mechanism of immune evasion in response to A2aR blockade. Critically, co-blockade of A2aR and TIM3 produces complete and durable tumour regression.ConclusionExpression of TIM3 represents a key mechanism of tumour-mediated immune evasion in response to A2aR blockade. The fact that co-blockade of A2aR and TIM3 mediates complete and durable tumour regression, with no obvious adverse effects, strongly supports the use of such dual blockade in the treatment of cancer.

Expression of multiple immune checkpoint molecules on T cells in malignant ascites from epithelial ovarian carcinoma.

Expression of immune checkpoint molecules, including programmed cell death protein-1 (PD-1), has been reported on T cells in various types of cancer. However, the expression status of these molecules in the tumor microenvironment of epithelial ovarian cancer (EOC) has not yet been studied. A total of 54 cases of malignant ascites from patients with EOC were analyzed in the present study. The expression of PD-1, lymphocyte-activation gene-3 (LAG-3), T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) and B and T lymphocyte attenuator (BTLA) on cluster of differentiation (CD)4+ and CD8+ T cells in malignant EOC ascites were investigated using multicolor flow cytometric analysis. The expression of PD-L1 in tumor cells, PD-L2 in HLA-DR-positive cells and galectin-9 in ascitic fluid was also analyzed. In addition, cytokine profiling of ascitic fluid was performed to understand the immune microenvironment of EOC. PD-1, LAG-3 TIM-3, and BTLA were expressed on 65.8, 10.6, 4.3 and 37.6% of CD4+ T cells, and on 57.7, 5.0, 4.9 and 15.7% of CD8+ T cells, respectively. Programmed cell death protein-1 (PD-1), LAG-3 and BTLA were more frequently expressed on CD4+ compared with CD8+ T cells. The co-expression of immune checkpoints was further investigated and results indicated that 39 (72.2%) and 37 patients (68.5%) expressed multiple immune checkpoints on CD4+ T cells and CD8+ T cells, respectively. In addition, lower levels of TNF-α and interleukin-6 in ascitic fluid were significantly associated with multiple immune checkpoint expression on CD8+ T cells. The present findings indicated that multiple immune checkpoint molecules were expressed on T cells in the EOC tumor microenvironment and the results may suggest the significance of simultaneous blockade of immune checkpoints to control EOC.

Recovery of hepatitis C specific T-cell responses after rituximab therapy in hepatitis C mixed cryoglobulinemic vasculitis.

Mixed cryoglobulinemic vasculitis is associated with monoclonal B cell expansion in patients with chronic hepatitis C (HCV) infection. B cell depletion therapy using rituximab, a CD20 monoclonal antibody, has been successful in achieving remission from symptomatic disease. This study investigated whether B cell depletion therapy has an impact on activation of HCV-specific T cell phenotype and function. Nineteen patients with Hepatitis C mixed cryoglobulinemic vasculitis were treated with 4 cycles of rituximab (375 mg/m2 ) and variables were measured 6 months after therapy. Using flow cytometry and Enzyme-Linked Immunospot assay, the number of activated and tissue-like B cells and number of T cells expressing Programmed cell death protein 1 (PD-1), T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), and multiple cytokines were measured before and after rituximab therapy. B cell depletion therapy is associated with a significant (P < 0.0001) decline in peripheral T cells with exhaustive phenotype, from pre-therapy to post-therapy-of rituximab (mean ± standard error): CD4+ (16.9 ± 0.9% to 8.9 ± 1.0%) and CD8+ (6.8 ± 0.6% to 3.0 ± 0.5%) T cells expressing PD-1 and CD4+ (11.0 ± 1.0% to 6.1 ± 0.8%) and CD8+ (12.7 ± 0.7% to 6.4 ± 0.4%) T cells expressing TIM-3. In addition, there was a significantly higher percentage of peripheral CD8+ T cells responding to HCV peptide stimulation in vitro secreting IFN-γ (4.55 ± 0.3 to 9.6 ± 1.0 IFN-γ/106 PBMCs, P < 0.0001), and more than one cytokine (1.3 ± 0.1% to 3.8 ± 0.2%, P < 0.0001) after therapy compared to pre-therapy. B cell depletion therapy results in recovery of T cell exhaustion and function in patients with HCV cryoglobulinemic vasculitis.

Agonist OX40 immunotherapy improves survival in glioma-bearing mice and is complementary with vaccination with irradiated GM-CSF-expressing tumor cells.

Glioma immunotherapy is an active area of clinical investigation. Glioma-associated immunosuppression remains an obstacle to efficacious immunotherapy, and combination approaches are likely necessary for durable success. OX40 is a member of the tumor necrosis factor receptor superfamily that is upregulated on activated lymphocytes, ligation of which results in enhanced activity and may be active against cancer. We sought to confirm the efficacy of agonist anti-OX40 immunotherapy against glioma and hypothesized that it is complementary with irradiated whole tumor cell vaccination. GL261 tumor cells were implanted into the right frontal lobes of syngeneic mice, which were then treated with controls, agonist anti-OX40 monoclonal antibody, vaccination with subcutaneous injection of irradiated granulocyte macrophage colony stimulating factor (GM-CSF)-expressing GL261 cells (GVAX), or vaccination + agonist anti-OX40 therapy. Animals were followed for survival. On day 18, splenocytes were harvested for enzyme-linked immunosorbent spot analyses and brains were harvested for immunohistochemistry and flow cytometry analyses of infiltrating lymphocytes. Combination immunotherapy with GVAX and systemic agonist anti-OX40 monoclonal antibody improved survival by 14 days over controls (median survival 36 vs 22 days, P < 0.00005). Systemically, T helper cell type 1 (Th1) antitumor immunity was enhanced significantly by combination therapy. In the brain, combination immunotherapy increased the percentage of Th1 CD4+ T lymphocytes and reduced the fraction that were Th2. In the brain, vaccination improved the ratio of CD8+ to FoxP3+ T lymphocytes, while combination immunotherapy reversed intracranial T-lymphocyte exhaustion, reducing their coexpression of programmed cell death protein 1 (PD-1) and T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) as well as PD-1 and lymphocyte-activation gene 3 (LAG-3). Anti-OX40 immunotherapy is active against intracranial glioma and synergizes with GVAX. Vaccination and anti-OX40 immunotherapy are mechanistically complementary, particularly in the glioma microenvironment.

Abstract 1641: Dual antibody blockade of TIM3 and PD1 on NYESO1 redirected human T cells leads to augmented control of lung cancer tumors

Abstract Background: Immunotherapy using checkpoint blockade has demonstrated impressive, durable responses in select patients with solid malignancies. This is especially true for the blockade of programmed death 1 (PD1). Currently, efforts are focused on understanding non-response. One hypothesis is that multiple inhibitory receptors (IRs) are upregulated on hypofunctional tumor-infiltrating lymphocytes (TILs) and necessitates blocking multiple IRs to increase the response rate. We describe data from a unique xenograft model of human lung cancer where tumor-reactive human T cells become hypofunctional and upregulate multiple IRs including PD1 and TIM3 (T cell immunoglobulin and mucin-domain containing-3). Combining a single IV dose of tumor-reactive T cells with blockade of both PD1 and TIM3 led to greater control of tumor growth than combining it with blockade of either alone. Materials/Methods: The A549 human lung cancer cell line was transduced to stably express the cancer-testis antigen, NYESO1, expressed in the contact of HLA-A2 (A549-A2-ESO). 5x106 A549-A2-ESO cells were injected subcutaneously into the flanks NSG mice. Activated T cells from healthy donors were lentivirally transduced with a TCR targeting NYESO1 (Ly95). After two weeks when tumors were established and measured ~150mm3, the mice were randomly assigned to one of the following treatments: 1) untreated, 2) non-transduced (NTD) T cells plus anti-PD1 and anti-TIM3 Ab, 3) Ly95 T cells, 4) Ly95 T cells + anti-PD1 Ab, 5) Ly95 T cells + anti-TIM3 Ab, 6) Ly95 T cells + anti-PD1 and anti-TIM3 Ab. T cells were injected IV at a single dose of 10x106/mouse. Abs were injected intraperitonealy (IP) at 10mg/kg every 5 days from the time of T cell injection. Tumor volumes were measured serially. At the end, the mice were sacrificed and the tumors were harvested, digested, and processed into single cell suspension. Flow cytometry was performed to look at degree of TIL infiltration and expression of surface markers. Ficoll gradient was used to isolate the T cells to conduct functional analyses. Results/Conclusion: By twenty days post T cell injection, the tumors in the Ly95 T cell group (3) were ~30% smaller than that of the untreated (1) and the NTD T cell + Ab group (2). Anti-PD1 Ab augmented Ly95 T cell control of tumor (4) (further decrease in size by ~30%) (4). Anti-TIM3 Ab had no effect on Ly95 T cell tumor control (5). Anti-PD1 plus anti-TIM3 Abs had the greatest augmentation on Ly95 T cell control of tumor size (6) (greater than 50% reduction in tumor size compared Ly95 T cells alone) The greatest TIL infiltration and ex-vivo TIL function were seen in tumors that received Ly95 T cell + anti-PD1 and anti-TIM3 Abs. Flow cytometry analysis revealed dynamic, adaptive upregulation of one IR when another IR was blocked. This explained why augmentation of Ly95 T cell control of tumor was greatest with combinatorial Ab blockade. Citation Format: Edmund Moon, Soyeon Kim, Shaun O'Brien, Naomi Saint Jean, Raluca Verona, Linda Snyder, Yangbing Zhao, Steven Albelda. Dual antibody blockade of TIM3 and PD1 on NYESO1 redirected human T cells leads to augmented control of lung cancer tumors [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 1641. doi:10.1158/1538-7445.AM2017-1641

Circulating biomarkers and outcomes in advanced non-small cell lung cancer patients treated with anti-PD1 (program death 1 receptor) monoclonal antibodies.

e20592 Background: Current evaluation of immunohistochemical expression of PDL-1 (program death receptor 1 ligand) can select some non-small cell lung cancer (NSCLC) patients who may benefit from anti-PD1 directed therapy. It is an imperfect marker and there is little information about systemic modulation of the immune system on therapy. In this study we explored the prognostic value of baseline circulating immune checkpoint and inflammatory molecules in advanced non-small cell lung cancer (NSCLC) patients receiving anti-PD1 therapy. Methods: Prospectively collected serum from advanced NSCLC patients receiving nivolumab or pembrolizumab were evaluated with the MILLIPLEX Human High Sensitivity T-cell (17-plex) and ProcartaPlex Human Immuno-Oncology Checkpoint (14-plex) panels on our Luminex FlexMAP 3D. Biomarker level cutoffs were optimized and evaluated against progression-free survival (PFS) and overall survival (OS) using log-rank analysis. Results: 21 cases were enrolled in this pilot study: 72% Caucasian, 61% female, 24% never-smokers. IL-10 was found to have a significant association with both PFS (p = 0.0055) and OS (p = 0.024), with levels below 3.32 pg/mL being associated with a superior clinical outcome. We also found IL-2 and IL-6 to have significant associations with PFS (p = 0.033 and 0.040, respectively), again, with low levels being associated with a superior outcome. Neither of these had significant associations with OS. Low circulating levels of the T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) protein were associated with superior PFS (p = 0.036), and a weak trend (p = 0.19) for OS. Conclusions: In this small exploratory pilot study we identified several circulating molecules associated with inflammation and immune system regulation that may have prognostic value for anti-PD-1 therapy. Notably, TIM-3 is a Th1-specific protein associated with macrophage activation and is also a component of T-cell exhaustion along with LAG3 and PD-1. Additional studies to follow up on these findings in larger cohorts are underway.

Significance of immune checkpoint proteins in EGFR-mutant non-small cell lung cancer

ObjectivesTo characterize the expression of PD-L1, PD-1, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and T-cell immunoglobulin and mucin-domain containing-3 (TIM3) in epidermal growth factor receptor (EGFR) mutant non-small cell lung cancer (NSCLC). Materials and methodsSamples from 90 patients with newly diagnosed advanced stage NSCLC harboring EGFR mutations and treated with first line EGFR tyrosine kinase inhibitors (TKI) within 3 months of diagnosis were stained for CTLA-4, PD-L1, PD-1, TIM-3 and CD3 expression by immunohistochemistry. ResultsPD-L1 was present in at least 1% of immune and tumor cells in 44% and 59% of samples, respectively. In multivariate analysis, increased CD3 immune shaped cell (ISC) counts (HR 2.805, p=0.034) and high PD-L1 tumor H-score (HR 3.805, p=0.022) was associated with a shorter progression free survival and high CTLA-4 ISC counts was associated with borderline overall survival significance (HR 1.054, p=0.061). ConclusionTumor PD-L1 expression was significantly associated with a shorter PFS whereas immune cell CTLA-4 may be prognostic for OS. Our findings support the ongoing development of CTLA-4 and PD1/PD-L1 inhibitors in this important molecularly defined subset of lung adenocarcinoma.

Circulating T lymphocyte subsets, cytokines, and immune checkpoint inhibitors in patients with bipolar II or major depression: a preliminary study

This study aimed to investigate the less known activation pattern of T lymphocyte populations and immune checkpoint inhibitors on immunocytes in patients with bipolar II disorder depression (BD) or major depression (MD). A total of 23 patients with BD, 22 patients with MD, and 20 healthy controls (HCs) were recruited. The blood cell count of T lymphocyte subsets and the plasma level of cytokines (IL-2, IL-4, IL-6, IL-10, TNF-α, and IFN-γ) were selectively investigated. The expression of T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), programmed cell death protein 1 (PD-1) and its ligands, PD-L1 and PD-L2, on T lymphocytes and monocytes, was detected. In results, blood proportion of cytotoxic T cells significantly decreased in BD patients than in either MD patients or HCs. The plasma level of IL-6 increased in patients with BD and MD. The expression of TIM-3 on cytotoxic T cells significantly increased, whereas the expression of PD-L2 on monocytes significantly decreased in patients with BD than in HCs. These findings extended our knowledge of the immune dysfunction in patients with affective disorders.