Restored T-cell Proliferation Research Articles

349 (PB129) - Novel, brain penetrant small molecule inhibitor of PD-L1 for targeting glioblastoma and brain metastasis

Background: The PD-1/PD-L1 molecular pathway is one of the primary mechanisms of immune evasion deployed by cancer cells and anti-PD-L1 monoclonal antibodies (mAbs) restore T-cell proliferation and enhanced tumor cell killing which has been shown to result in clinically revolutionary efficacy in many tumor types. Unfortunately, this is not the case in CNS tumors where these mAbs have failed to show improved responses and survival. It appears that one of the main reasons for this is the poor brain penetrance of these mAbs and clinical evidence suggests that increasing brain penetrance results in better efficacy.

P392: PHARMACOLOGICAL INHIBITION OF SYK CONFERS ANTI-PROLIFERATIVE AND NOVEL ANTI-TUMOR IMMUNE RESPONSES IN AML

Background: Spleen tyrosine kinase (SYK) acts as a key integrator of signals from cell surface receptors containing an immunoreceptor tyrosine-based activation motif to boost cellular proliferation. In acute myeloid leukemia (AML), SYK serves as a relay to an oncogenic transcriptional regulatory network linked to NPM1, HOXA9 and MEIS1. The selective, orally bioavailable SYK inhibitor entospletinib (ENTO) has demonstrated clinical activity and tolerability in HOXA9/MEIS1-driven AML. ENTO is currently being investigated in a global Phase 3 trial, AGILITY (NCT05020665), in combination with intensive induction/consolidation chemotherapy in patients (pts) with treatment-naive NPM1-mutated (NPM1m) AML. Lanraplenib (LANRA) is a next-generation SYK inhibitor with similar potency and selectivity to ENTO but with more favorable pharmacologic properties that is currently being evaluated in combination with gilteritinib in pts with relapsed or refractory FLT3-mutated AML (NCT05028751). Aims: To investigate the effects of ENTO and LANRA in T-cell responses in AML. Methods: All pt-derived bone marrow (BM) and peripheral blood samples were obtained in accordance with IRB (OHSU#004422) approval. Results from gene expression profiling studies (RNA sequence [RNA-seq]) in bone marrow/peripheral blood mononuclear cells (MNCs) derived from 152 pts with AML, for which matching NPM1 status and ex vivo sensitivity to ENTO was available, previously published by Tyner et al (2018), were reanalyzed using trimmed mean of M values normalization and differential expression using edgeR. ENTO/LANRA sensitivity was assessed ex vivo after 72 hours in culture at 37°C by MTS assay. T-cell functional assays were performed by culturing MNCs with anti-CD3 and treating with ENTO or LANRA, singly and in combination with anti-PD1. CD3+ T-cell proliferation as measured by Ki67 expression and pSYK activation were assessed by quantitative single-cell immunofluorescence microscopy. Six formalin-fixed, paraffin-embedded BM biopsies from newly diagnosed AML pts were assessed for spatial expression of mRNA using a digital spatial profiling method. Results: In AML, mutations in NPM1 with co-expression of HOXA9/MEIS1 at baseline predicted anti-proliferative activity to ENTO in ex vivo drug sensitivity studies. Accordingly, treatment of leukemic cells with either ENTO or LANRA inhibited SYK auto-phosphorylation in a dose-dependent manner. Pathway analysis of archival RNA-seq data from pts enrolled in the Leukemia and Lymphoma Society BEAT AML Master Protocol (NCT03013998 [BAML-16-001-S6]) revealed gene expression signatures at baseline associated with the observed sensitivity to ENTO, including significant enrichment in the expression of genes associated with leukemogenesis, myeloid differentiation and immune regulation. This was supported by T-cell functional studies, which demonstrated that ENTO and LANRA, singly as well as in combination with anti-PD1, could restore T-cell proliferation in primary AML pt samples that exhibited suppression. Lastly, Cancer Transcriptome Atlas analysis on archival BM biopsies obtained from these AML pts showed a strong correlation between immune checkpoint response and gene expression associated with immune pathway signaling. Summary/Conclusion: Inhibition of SYK is a promising therapeutic approach in NPM1m AML. Our studies illustrate that ENTO and LANRA may restore T-cell proliferation in a subset of AML pts with dysfunctional T-cell responses, suggesting a novel mechanism of action. Additional studies are required to fully understand the mechanism of SYK-mediated antitumor immune responses in AML.

Targeting CD73 with AB680 (Quemliclustat), a Novel and Potent Small-Molecule CD73 Inhibitor, Restores Immune Functionality and Facilitates Antitumor Immunity.

T cells play a critical role in the control of cancer. The development of immune checkpoint blockers (ICB) aimed at enhancing antitumor T-cell responses has revolutionized cancer treatment. However, durable clinical benefit is observed in only a subset of patients, prompting research efforts to focus on strategies that target multiple inhibitory signals within the tumor microenvironment (TME) to limit tumor evasion and improve patient outcomes. Adenosine has emerged as a potent immune suppressant within the TME, and CD73 is the major enzyme responsible for its extracellular production. CD73 can be co-opted within the TME to impair T-cell-mediated antitumor immunity and promote tumor growth. To target this pathway and block the formation of adenosine, we designed a novel, selective, and potent class of small-molecule inhibitors of CD73, including AB680 (quemliclustat), which is currently being tested in patients with cancer. AB680 effectively restored T-cell proliferation, cytokine secretion, and cytotoxicity that were dampened by the formation of immunosuppressive adenosine by CD73. Furthermore, in an allogeneic mixed lymphocyte reaction where CD73-derived adenosine had a dominant suppressive effect in the presence of PD-1 blockade, AB680 restored T-cell activation and function. Finally, in a preclinical mouse model of melanoma, AB680 inhibited CD73 in the TME and increased the antitumor activity of PD-1 blockade. Collectively, these data provide a rationale for the inhibition of CD73 with AB680 in combination with ICB, such as anti-PD-1, to improve cancer patient outcomes.

Modulation of Xenogeneic T-cell Proliferation by B7 and mTOR Blockade of T Cells and Porcine Endothelial Cells.

Activation of porcine endothelial cells (PECs) is the mechanistic centerpiece of xenograft rejection. This study sought to characterize the immuno-phenotype of human T cells in response to PECs and to explore the immuno-modulation of B7 and mammalian target of rapamycin blockade of T cells and/or PECs during xeno-responses. Rapid memory T-cell (TM) responses to PECs were assessed by an intracellular cytokine staining. T-cell proliferation to PEC with or without belatacept or rapamycin was evaluated by a mixed lymphocyte-endothelial cell reaction (MLER). Additionally, rapamycin-pretreated PECs were used in MLER. Cell phenotypes were analyzed by flow cytometry. Tumor necrosis factor-α/interferon-γ producers were detected in CD8+ cells stimulated by human endothelium but not PECs. MLER showed proliferation of CD4+ and CD8+ cells with predominantly memory subsets. Purified memory and naive cells proliferated following PEC stimulation with an increased frequency of TM in PEC-stimulated naive cells. Proliferating cells upregulated programmed cell death-1 (PD-1) and CD2 expression. Belatacept partially inhibited T-cell proliferation with reduced CD2 expression and frequency of the CD8+CD2highCD28- subset. Rapamycin dramatically inhibited PEC-induced T-cell proliferation, and rapamycin-preconditioned PECs failed to induce T-cell proliferation. PD-1 blockade did not restore T-cell proliferation to rapamycin-preconditioned PECs. Humans lack rapid TM-mediated responses to PECs but induce T-cell proliferative responses characterized largely as TM with increasing CD2 and PD-1 expression. B7-CD28 and mammalian target of rapamycin blockade of T cells exhibit dramatic inhibitory effects in altering xeno-proliferating cells. Rapamycin alters PEC xeno-immunogenicity leading to inhibition of xeno-specific T-cell proliferation independent of PD-1-PD ligand interaction.

Abstract 1630: Novel, small molecule inhibitors of PD-L1/PD-1 interaction

Abstract Introduction: The PD-1/PD-L1 molecular pathway is one of the primary mechanisms of immune evasion deployed by cancer cells. Induction of PD-L1 expression on cancer cells is associated with inhibition of immune responses against cancer, thus favouring cancer progression and metastasis. Activation of PD-1/PD-L1 pathway induces T-cell anergy and exhaustion and enhances the function of regulatory T-cells (Tregs) thereby leading to an immune suppressive environment. Therefore, blocking this pathway restores the proliferation and cytotoxicity of T- lymphocytes, inhibits the function of Tregs and results in decreased T-cell apoptosis. A number of monoclonal antibodies agents targeting PD-1/PD-L1 have approved for a number of malignancies. These approved therapies require bolus intravenous injections, are administered in high dose and have a long half-life. The long residence time of these mAbs could contribute to the well-documented drug-related adverse effects. Therefore, there is still a need for potent, selective small molecule inhibitors of the PD-1/PD-L1 pathway. Such small molecule inhibitors, can provide increased oral bioavailability, and shorter half-life activity for a more controllable treatment, and flexibility for combination strategies. Methods: Rational design approaches were used to design novel small molecule PD-1/PD-L1 pathway inhibitors; potency of these inhibitors was assessed in an in-vitro TR-FRET assay. Checkpoints signaling reporter assays as well as ex-vivo co-culture assays were used to assess the ability of the compounds to restore T-cell proliferation and function. In vivo efficacy was assessed by syngeneic and humanized models in mice. Results: Our lead molecule JTi showed strong in vitro IC50 of 0.8 nM in TR-FRET assay that measures interaction between PD-1 and PD-L1. This molecule also augmented T-cell response as measured by IFN-gamma activation in a cancer cell-PBMC based co-culture assay. This molecule induced dimerization of PD-L1 in U2OS cell based assay with an EC50 of ~300 nM. Competition study between anti-PD-L1 blocking antibody and this small molecule inhibitor clearly suggested that it binds at the same site as the antibody. In a cell based assay, measuring PD-1/PD-L1 interaction, JTi clearly demonstrated inhibition of PD-1 and PD-L1 binding in a dose-dependent manner. JTi showed comparable efficacy to the anti-PD-L1 antibody in syngeneic (4T1, CT-26) and humanized models (MC-38/hPD-L1) by oral administration. Conclusion: Advanced studies to further characterize and progress the molecule into pre-clinical development are in progress. The oral administration route of these small molecule PD-1/PD-L1 inhibitors would provide an attractive option to be used as a maintenance therapy followed by mAb based treatment to enhance patient compliance. The lead compound will be entering IND enabling studies in the 1st half of 2021. Citation Format: Dhanalakshmi Sivanandhan, Sridharan Rajagopal, Naveen Sadhu M, Chandru Gajendran, Chandregowda venkateshappa, Muralidhar Reddy, Pendyala Satya Kishore, Pratima Deshpande, Sundarajan kannan, Tabassum Sahareen, Santhosh Viswakarma, Amir Siddiqui, Mohammed Zainuddin, Rudresh G, Prashanthi Daram, Ramchandraiah Gosu, Rashmi Rekha Devi. Novel, small molecule inhibitors of PD-L1/PD-1 interaction [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 1630.

Preclinical Characterization of Linrodostat Mesylate, a Novel, Potent, and Selective Oral Indoleamine 2,3-Dioxygenase 1 Inhibitor.

Tumors can exploit the indoleamine 2,3-dioxygenase 1 (IDO1) pathway to create an immunosuppressive microenvironment. Activated IDO1 metabolizes tryptophan into immunosuppressive kynurenine, leading to suppressed effector T-cell (Teff) proliferation, allowing for tumor escape from host immune surveillance. IDO1 inhibition counteracts this immunosuppressive tumor microenvironment and may improve cancer outcomes, particularly when combined with other immunotherapies. Linrodostat mesylate (linrodostat) is a potent, selective oral IDO1 inhibitor that occupies the heme cofactor-binding site to prevent further IDO1 activation and is currently in multiple clinical trials for treatment of patients with advanced cancers. Here, we assess the in vitro potency, in vivo pharmacodynamic (PD) activity, and preclinical pharmacokinetics (PKs) of linrodostat. Linrodostat exhibited potent cellular activity, suppressing kynurenine production in HEK293 cells overexpressing human IDO1 and HeLa cells stimulated with IFNγ, with no activity against tryptophan 2,3-dioxygenase or murine indoleamine 2,3-dioxygenase 2 detected. Linrodostat restored T-cell proliferation in a mixed-lymphocyte reaction of T cells and allogeneic IDO1-expressing dendritic cells. In vivo, linrodostat reduced kynurenine levels in human tumor xenograft models, exhibiting significant PD activity. Linrodostat demonstrated a PK/PD relationship in the xenograft model, preclinical species, and samples from patients with advanced cancers, with high oral bioavailability in preclinical species and low to moderate systemic clearance. Our data demonstrate that linrodostat potently and specifically inhibits IDO1 to block an immunosuppressive mechanism that could be responsible for tumor escape from host immune surveillance with favorable PK/PD characteristics that support clinical development.

Contribution of long-chain fatty acid to induction of myeloid-derived suppressor cell (MDSC)-like cells – induction of MDSC by lipid vesicles (liposome)

Context Effects of liposomal particles on immune function have not been adequately investigated. Earlier reports indicate that intravenous injection of rats with pegylated liposomes comprising chemically defined specific lipids produces myeloid derived suppressor-cell (MDSC)-like cells in the spleen. Objectives After liposome injection, we sought a cell surface marker expressed specifically on splenic macrophages. Then we assessed the immunosuppressive activity of macrophages positive for the marker. Furthermore, we investigated whether immunosuppression induction is an immunopharmacological action specific to this pegylated liposome, or not. Materials and Methods After using a microarray system to screen genes enhanced by this liposome, we evaluated cell surface expression of gene products using flow cytometry. Liposomes of several kinds, each comprising one type of phospholipid, were prepared and evaluated for their ability to induce T-cell suppression. Results Microarray analysis indicated enhanced B7-H3 expression. Flow cytometry revealed that the B7-H3 molecule was expressed on splenic macrophages after liposome injection. B7-H3+ macrophages were positive for iNOS. Removing B7-H3+ cells restored T-cell proliferation. Similarly to this liposome, various liposomes with different long chain fatty acids induced T-cell suppression when accumulated in the spleen. Conclusions Immunosuppressive cells induced by this pegylated liposome closely resemble MDSCs, especially B7-H3+ MDSCs. Immunosuppression induction is not a phenomenon specific to this liposome. Accumulation of long chain fatty acid in macrophages by internalization of liposomal nanoparticles might be related to macrophage acquisition of immunosuppressive activity in vivo.

IFN-α Suppresses Myeloid Cytokine Production, Impairing IL-12 Production and the Ability to Support T-Cell Proliferation.

Interferon-α (IFN-α) can suppress production of T-cell polarizing cytokines or induce inhibitory antigen-presenting cells that suppress T-cell activation. Previous studies showed that IFN-α therapy fails to boost virus-specific T-cell immunity in patients with chronic hepatitis B virus infection. Our aim was to determine whether IFN-α exposure alters human antigen-presenting cell function in vivo. We investigated the immunomodulatory effects using peripheral blood mononuclear cells from healthy donors exposed to IFN-α and chronic hepatitis B (CHB) patients starting IFN-α therapy. IFN-α increased HLA-DR, CD80, CD86, and PD-L1 expression on healthy donor monocytes. In contrast to the activated phenotype, IFN-α inhibited Toll-like receptor-induced cytokine production and monocyte-induced T-cell proliferation. In CHB patients, peg-IFN treatment induced an interferon-stimulated gene signature in monocytes and increased HLA-DR, CD80, CD86, and PD-L1 expression. As early as 3 days after CHB patients started treatment, IFN-α inhibited monocyte cytokine production and T-cell stimulation ex vivo. IFN-α-mediated inhibition of IL-12 production, rather than inhibitory receptor expression, was responsible for inhibition of T-cell proliferation. Addition of IL-12 restored T-cell proliferation to baseline levels. Understanding how professional antigen-presenting cells respond to immunomodulation is important for both new innate and adaptive-targeted immunotherapies. NCT00962871.

Acute myeloid leukemia–induced T-cell suppression can be reversed by inhibition of the MAPK pathway

AbstractAcute myeloid leukemia (AML) remains difficult to treat due to mutational heterogeneity and the development of resistance to therapy. Targeted agents, such as MEK inhibitors, may be incorporated into treatment; however, the impact of MEK inhibitors on the immune microenvironment in AML is not well understood. A greater understanding of the implications of MEK inhibition on immune responses may lead to a greater understanding of immune evasion and more rational combinations with immunotherapies. This study describes the impact of trametinib on both T cells and AML blast cells by using an immunosuppressive mouse model of AML and primary patient samples. We also used a large AML database of functional drug screens to understand characteristics of trametinib-sensitive samples. In the mouse model, trametinib increased T-cell viability and restored T-cell proliferation. Importantly, we report greater proliferation in the CD8+CD44+ effector subpopulation and impaired activation of CD8+CD62L+ naive cells. Transcriptome analysis revealed that trametinib-sensitive samples have an inflammatory gene expression profile, and we also observed increased programmed cell death ligand 1 (PD-L1) expression on trametinib-sensitive samples. Finally, we found that trametinib consistently reduced PD-L1 and PD-L2 expression in a dose-dependent manner on the myeloid population. Altogether, our data present greater insight into the impact of trametinib on the immune microenvironment and characteristics of trametinib-sensitive patient samples.

Ligustilide inhibits the activation of cancer-associated fibroblasts

The purpose of this work was to study the effects and underlying molecular mechanisms of ligustilide on cancer-associated fibroblasts (CAFs). The effects of ligustilide on the growth of CAFs and splenocytes were detected by MTT assay, and flow cytometry was used to detect effects on T-cell proliferation. Western blotting was used to detect the expression levels of CAF-related proteins after ligustilide treatment. This study found that ligustilide had no effect on the growth of splenocytes but that it could change the immunosuppressive function of CAFs through the TLR4-NF-κB pathway and restore T-cell proliferation previously inhibited by the CAF supernatant. Thus, ligustilide is expected to be a candidate for new antitumor drugs.

Abstract 5552: Novel, heterocyclic small molecule inhibitors of PD-1 and PD-L1 pathway

Abstract The PD-1/PD-L1 molecular pathway is one of the primary mechanisms of immune evasion deployed by cancer cells. Induction of PD-L1 expression on cancer cells is associated with inhibition of immune responses against cancer, thus permitting cancer progression and metastasis. Activation of PD-1/PD-L1 pathway induces apoptosis of activated T-cells, inhibits their proliferation, facilitates T-cell anergy and exhaustion and enhances the function of regulator T-cells. Therefore, blocking this pathway restores the proliferation and cytotoxicity of CTLs, inhibits the function of Tregs and results in decreased T-cell apoptosis. A number of cancer immunotherapy agents targeting PD-1/PD-L1 have been developed and approved for a number of malignancies (PD-1: Nivolumab, Pembrolizumab, PD-L1: Atezolizumab, Avelumab, Durvalumab). However, there is a still a need for potent, selective small molecule inhibitors of the PD-1/PD-L1 pathway. The approved therapies require bolus intravenous injections, are administered in high dose and have a long half life. The long residence time of these mAbs could contribute to the well-documented drug-related adverse effects. Small molecule inhibitors, therefore, can provide increased oral bioavailability, increased bio-efficiency and shorted half life activity for a more controllable treatment, particular in the case of auto-immune or other adverse effects. Rational and structure guided de novo design approaches were used to design novel small molecule PD-1/PD-L1 pathway inhibitors; potency of these inhibitors was assessed in an in-vitro TR-FRET assay. Checkpoints signaling reporter assays as well as ex-vivo co-culture assays were used to assess the ability of the compounds to restore T-cell proliferation and function. Three novel chemical series as potent PD-1/PD-L1 pathway inhibitors are being developed for the treatment of cancer. Compounds from these series showed strong in vitro potency of 0.01 to 0.2 µM against PD-1/PD-L1. JBI-426 exhibited an IC50 of 0.04 µM and no cytotoxicity against cancer cell proliferation per se. JBI-426 showed good in vitro ADME properties in terms of aqueous solubility, metabolic stability, permeability and excellent oral bioavailability in mouse pharmacokinetics. In a RENCA syngeneic model, oral administration of JBI-426 at 50 mg/kg resulted in a strong tumor growth inhibition, comparable (or better) than the PD-L1 mAb, and was well tolerated. The effect of JBI-426 on tumor infiltrating lymphocytes was also assessed; a significant increase in CD8+ cytotoxic lymphocytes was observed. Further studies to assess additional compounds from the three chemical series are underway. The oral administration route of these PD-1/PD-L1 inhibitors would provide an attractive alternate to the currently available antibodies in treating cancer either as a stand-alone therapy or in combination with other immuno-modulatory agents, as well as other standard of care agents. Citation Format: Sivanandhan Dhanalakshmi, Shivani Garapaty, Chandregowda Venkateshappa, Guru Pavan Seerapu, Reshma Das, Pradeep Nagaraj, Ronodip Kar, Anuj Kumar Singh, Venkatesha Ashokkumar Venkatesubbiah, Ramakishore VP Putta, Muralidhar Pendyala, Girisha Lokesh, Hari Madaka, Harikrishna Reddy Thummuru, Shikas AP, Prateeksha Anchan, Prathima Bhat, Rudresha G, Mohd Zainuddin, Krishnakumar V, Ramachandraiah Gosu, Rajendra Kristam, Jeyaraj DA, Sriram Rajagopal. Novel, heterocyclic small molecule inhibitors of PD-1 and PD-L1 pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5552.

Tumor-Derived GM-CSF Promotes Granulocyte Immunosuppression in Mesothelioma Patients.

Purpose: The cross-talk between tumor cells, myeloid cells, and T cells can play a critical role in tumor pathogenesis and response to immunotherapies. Although the etiology of mesothelioma is well understood, the impact of mesothelioma tumor cells on the surrounding immune microenvironment is less well studied. In this study, the effect of the mesothelioma tumor microenvironment on circulating and infiltrating granulocytes and T cells is investigated.Experimental Design: Tumor tissues and peripheral blood from mesothelioma patients were evaluated for presence of granulocytes, which were then tested for their T-cell suppression potential. Different cocultures of granulocytes and/or mesothelioma tumor cells and/or T cells were set up to identify the mechanism of T-cell inhibition.Results: Analysis of human tumors showed that the mesothelioma microenvironment is enriched in infiltrating granulocytes, which inhibit T-cell proliferation and activation. Characterization of the whole blood at diagnosis identified similar, circulating, immunosuppressive CD11b+CD15+HLADR- granulocytes at increased frequency compared with healthy controls. Culture of healthy-donor granulocytes with human mesothelioma cells showed that GM-CSF upregulates NOX2 expression and the release of reactive oxygen species (ROS) from granulocytes, resulting in T-cell suppression. Immunohistochemistry and transcriptomic analysis revealed that a majority of mesothelioma tumors express GM-CSF and that higher GM-CSF expression correlated with clinical progression. Blockade of GM-CSF with neutralizing antibody, or ROS inhibition, restored T-cell proliferation, suggesting that targeting of GM-CSF could be of therapeutic benefit in these patients.Conclusions: Our study presents the mechanism behind the cross-talk between mesothelioma tumors and the immune microenvironment and indicates that targeting GM-CSF could be a novel treatment strategy to augment immunotherapy in patients with mesothelioma. Clin Cancer Res; 24(12); 2859-72. ©2018 AACR.

Abstract 4964: Structure, in vitro biology and in vivo pharmacodynamic characterization of a novel clinical IDO1 inhibitor

Abstract The enzyme indoleamine 2,3-dioxygenase 1 (IDO1) catalyzes the degradation of tryptophan along the kynurenine pathway, and is frequently expressed in human malignancies. The activity of IDO1 induces an immunosuppressive microenvironment in tissues by inhibiting T-cell function through local depletion of tryptophan and through generation of kynurenine pathway metabolites. Inhibition of IDO1 is expected to diminish the immunosuppressive tumor microenvironment and improve cancer patient outcomes, particularly when used in combination with cancer immunotherapy agents such as nivolumab and ipilimumab. In this presentation, we will disclose the chemical structure, enzyme inhibitory mechanism, in vitro potency and in vivo pharmacodynamic (PD) activity of BMS’ IDO1 inhibitor currently in Phase I clinical trials. The compound is a potent and selective IDO1 inhibitor with no activity against another tryptophan degrading enzyme, tryptophan 2,3-dioxygenase (TDO). It exhibited potent cellular activity, suppressing kynurenine production in HEK293 cells overexpressing human IDO1 (IC50 = 1.1 nM) and in HeLa cells stimulated with IFNγ (IC50 = 1.7 nM). The compound also potently restored T-cell proliferation in a co-culture of T cells and human cancer cells and in a mixed lymphocyte reaction where T cells were co-cultured with allogeneic IDO1-expressing dendritic cells (EC50 = 1.2 nM). In vivo, when given once a day orally, the compound exhibited significant PD activity in mouse tumors grown subcutaneously in syngeneic hosts and in human tumors grown as xenografts in nude mice. Citation Format: John T. Hunt, Aaron Balog, Christine Huang, Tai-An Lin, Tai-An Lin, Derrick Maley, Johnni Gullo-Brown, Jesse Swanson, Jennifer Brown. Structure, in vitro biology and in vivo pharmacodynamic characterization of a novel clinical IDO1 inhibitor [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 4964. doi:10.1158/1538-7445.AM2017-4964

Systemic T Cells Immunosuppression of Glioma Stem Cell-Derived Exosomes Is Mediated by Monocytic Myeloid-Derived Suppressor Cells.

A major contributing factor to glioma development and progression is its ability to evade the immune system. Nano-meter sized vesicles, exosomes, secreted by glioma-stem cells (GSC) can act as mediators of intercellular communication to promote tumor immune escape. Here, we investigated the immunomodulatory properties of GCS-derived exosomes on different peripheral immune cell populations. Healthy donor peripheral blood mononuclear cells (PBMCs) stimulated with anti-CD3, anti-CD28 and IL-2, were treated with GSC-derived exosomes. Phenotypic characterization, cell proliferation, Th1/Th2 cytokine secretion and intracellular cytokine production were analysed by distinguishing among effector T cells, regulatory T cells and monocytes. In unfractionated PBMCs, GSC-derived exosomes inhibited T cell activation (CD25 and CD69 expression), proliferation and Th1 cytokine production, and did not affect cell viability or regulatory T-cell suppression ability. Furthermore, exosomes were able to enhance proliferation of purified CD4+ T cells. In PBMCs culture, glioma-derived exosomes directly promoted IL-10 and arginase-1 production and downregulation of HLA-DR by unstimulated CD14+ monocytic cells, that displayed an immunophenotype resembling that of monocytic myeloid-derived suppressor cells (Mo-MDSCs). Importantly, the removal of CD14+ monocytic cell fraction from PBMCs restored T-cell proliferation. The same results were observed with exosomes purified from plasma of glioblastoma patients. Our results indicate that glioma-derived exosomes suppress T-cell immune response by acting on monocyte maturation rather than on direct interaction with T cells. Selective targeting of Mo-MDSC to treat glioma should be considered with regard to how immune cells allow the acquirement of effector functions and therefore counteracting tumor progression.

OP15. ENHANCEMENT OF MYELOID DENDRITIC CELLS THROUGH MAPK P38 INHIBITION PROMOTES T CELL PROLIFERATION AND RESTORES ADAPTIVE IMMUNITY IN GBM PATIENTS

AbstractINTRODUCTION: Immunotherapy is an emerging backbone to oncology treatment in a number of cancers, mainly due to the advent of checkpoint blockade therapy. In Glioblastoma (GBM) immunotherapies such as peptide vaccines and laboratory generated monocyte-derived dendritic cells (moDC) are demonstrating promise in advanced stages of clinical trials. Recently however, a number of studies have highlighted the immune advantages circulating DC possess. We previously showed that the potential novel adoptive transfer of isolated circulating myeloid DC (MDC1) may be hampered in GBM patients due to reduced numbers of MDC1 and suppressed production of the key Th1-polarising cytokine IL-12. Importantly, IL-12 production by MDC1 was rescued and enhanced upon exposure to a MAPK p38 inhibitor (p38i). The increase in IL-12 with p38i was unique to MDC1s and in contrast to its effects on moDC. Here we tested the hypothesis that p38i enhances the capacity of MDC1 to promote T-cell proliferation. METHOD: Paramagnetic bead isolation kits were used to isolate circulating MDC1, CD4+ cells or peripheral blood lymphocytes (PBL) from mononuclear cells. A model of GBM:DC interaction was devised in which DC were co-cultured with tumour lysate (TL) from U87 (TL:DC ratio 50:1), or dexamethasone (Dex) 10-6M. The p38i, BIRB0796 1μM, was added to inhibit the MAPK pathway in MDC1s. The impact of GBM/Dex on DC function was determined in a mixed lymphocyte reaction (MLR) with PBL as responders. Further experiments, determined the impact of checkpoint inhibitors (Ipilimumab and Pembrolizumab) on the MLR. The MLR supernatants were analysed for IFN-γ and IL-10. RESULTS: Exposure to TL or Dex markedly inhibited the capacity of MDC1 to stimulate an MLR (p=0.001 and p<0.001 respectively). However, pre-treatment with p38i reversed this increasing proliferation above that of control cells (TL, p=0.001; DEX, p=0.021). Analysis of the supernatants of p38i-treated MDC1 stimulated PBL, showed a prominent increase in the Th1-cytokine IFN-γ. The addition of checkpoint inhibitors to TL or Dex treated MDC1 also restored T-cell proliferation. However, the greatest proliferation was observed with the combination of p38i MDC1s and checkpoint inhibitors. This combination was associated with further increases in IFN-γ secretion. CONCLUSION: MAPK p38 inhibited MDC1s stimulate an adaptive immune response greater than that of healthy MDC1s. Furthermore, this can reverse immunosuppression from Dex and TL on DCs, experienced by GBM patients. Interestingly, the combination of p38i MDC1 with checkpoint blockade inhibitors was most favourable at stimulating a lymphocyte response, postulating an advantageous immunotherapeutic strategy in patients.

Abstract A135: Novel immunotherapy for intractable pancreatic cancer focusing on the myeloid derived suppressor cells in the tumor microenvironment

Abstract Pancreatic cancer is one of malignant diseases with poor prognosis. Despite the recent progress for preoperative neoadjuvant chemoradiotherapy, the long term prognosis is still poor to make forward new treatment strategy. We have recently reported novel mechanism of anticancer reagents to impact patients' prognosis improving tumor microenvironment other than its direct cytotoxic effects (Ann Surg Oncol 2013, Cancer Sci 2014). The underlying mechanism include inhibition of HLA class I down regulation and Treg infiltration, increase of CD4 and CD8 T cell infiltration (Int J Clin Oncol 2015, Cancer Res 2015). Here we report that GM-CSF production was significantly enhanced in various PDAC cell lines after treatment with chemotherapy including gemcitabine or 5-FU, which induced the differentiation of CD14 positive monocytes into myeloid-derived suppressor cells (MDSC). This differentiation was promoted through MAPK signal pathway or NFkB pathway. Furthermore, blockade of GM-CSF with monoclonal antibodies helped to restore T-cell proliferation when co-cultured with monocytes stimulated with tumor supernatants. GM-CSF expression was also observed in primary tumors and correlated with poor prognosis in 68 PDAC patients analyzed by immunohistochemistry. We expect that postoperative prognosis of pancreatic cancer would be more improved by multidisciplinary strategy targeting MDSC and GM-CSF followed by curative surgical intervention. Citation Format: Takahiro Tsuchikawa, Shintaro Takeuchi, Toru Nakamura, Mizuna Takahashi, kazuho Inoko, Toshihiro Kushibiki, Koji Hontani, Masato Ono, Shota Kuwabara, Toshiaki Shichinohe, Satoshi Hirano. Novel immunotherapy for intractable pancreatic cancer focusing on the myeloid derived suppressor cells in the tumor microenvironment [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A135.

Chemotherapy-Derived Inflammatory Responses Accelerate the Formation of Immunosuppressive Myeloid Cells in the Tissue Microenvironment of Human Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic malignancies. PDAC builds a tumor microenvironment that plays critical roles in tumor progression and metastasis. However, the relationship between chemotherapy and modulation of PDAC-induced tumor microenvironment remains poorly understood. In this study, we report a role of chemotherapy-derived inflammatory response in the enrichment of PDAC microenvironment with immunosuppressive myeloid cells. Granulocyte macrophage colony-stimulating factor (GM-CSF) is a major cytokine associated with oncogenic KRAS in PDAC cells. GM-CSF production was significantly enhanced in various PDAC cell lines or PDAC tumor tissues from patients after treatment with chemotherapy, which induced the differentiation of monocytes into myeloid-derived suppressor cells (MDSC). Furthermore, blockade of GM-CSF with monoclonal antibodies helped to restore T-cell proliferation when cocultured with monocytes stimulated with tumor supernatants. GM-CSF expression was also observed in primary tumors and correlated with poor prognosis in PDAC patients. Together, these results describe a role of GM-CSF in the modification of chemotherapy-treated PDAC microenvironment and suggest that the targeting of GM-CSF may benefit PDAC patients' refractory to current anticancer regimens by defeating MDSC-mediated immune escape.

ITOC2 – 011. Identification of second-generation potent and selective inhibitors of indoleamine-2,3-dioxygenase-1 (IDO1) for the treatment of cancer

Background IDO1 inhibition with a 1st-generation inhibitor has shown promising clinical benefit in melanoma patients, as well as exacerbated toxicity, when combined with ipilimumab. We have identified a novel class of IDO1 inhibitors (IDO1i), which surpass the potency, selectivity and pharmacokinetic parameters of 1st-generation IDO1i. Methods IDO1 inhibition was assessed from kynurenine (kyn) levels produced by stimulated HeLa or dendritic cell cultures or recombinant IDO1. T-cell proliferation was measured using eFluor®670 dye. CT26 tumours were grown SC in Balb/c mice. IDO1i and kyn levels were measured by LC/MS/MS. Assessment of immune parameters was performed on tumour-infiltrating leucocytes by FACS. Findings These IDO1i potently inhibit cellular IDO1 with IC50 ≤ 10 nM, retain their potency in human serum (IC50 ≤ 10 nM) and restore T-cell proliferation, which is inhibited by allogeneic IDO1 + DC (EC50 = 2–5 nM). They exhibit PK characteristics consistent with once-daily dosing in humans. One of these molecules, selected for clinical evaluation, has undergone extensive characterisation, including the relationship between plasma levels of compound and kyn, and its impact on tumour growth and immune parameters in mouse syngeneic tumours, as single agent and in combination with various immune checkpoint inhibitors. Discussion This study provides the preclinical basis for clinical evaluation of a 2nd-generation IDO1i in combination with other immunotherapies.

Increased PD-L1 expression and PD-L1/CD86 ratio on macrophages infected with Neisseria gonorrhoeae contributes to the functional Suppression of T-cell responses.

Event Abstract Back to Event Increased PD-L1 expression and PD-L1/CD86 ratio on macrophages infected with Neisseria gonorrhoeae contributes to the functional Suppression of T-cell responses. Alejandro Escobar1*, Carolina Ortiz1, Killen Garcia1, Paula Rodas2 and Claudio Acuña-Castillo3 1 Universidad de Chile, Instituto de Investigación en Ciencias Odontológicas, Chile 2 Universidad Andres Bello, Center for Integrative Medicine and Innovative Science, Chile 3 Universidad de Santiago, Chile Neisseria gonorrhoeae is the etiological agent of the sexually transmitted disease gonorrhoea, which is widespread throughout the world. Infections with Neisseria gonorrhoeae do not improve immune response in patients with re-infection, suggesting that gonococcus displays several mechanisms to evade immune response and survive in the host. Our data indicate that Neisseria gonorrhoeae is able to suppress the protective immune response in macrophages, polarized them towards an immunoregulatory profile characterized by up-regulation of M2-Macrophage-associated markers and IL-10. Considering there are numerous reports indicating the capacity of Intracellular pathogens like Mycobacteria to impair innate and adaptative immune response using inhibitory role played by Programmed Death 1 and Programmed Death Ligand 1 (PD-1/PD-L1) interactions, in this study, we evaluated surface co-stimulatory (CD86, CD40) and co-inhibitory (PD-L1) markers expression and allostimulatory capacity in macrophages infected with Neisseria gonorrhoeae. Results showed that Neisseria gonorrhoeae was not able to induce the co-stimulatory CD86 neither CD40. However, we found a significant up-regulation of PD-L1 in macrophages upon gonococcal infection. PD-L1/CD86 ratio was increased and positively correlated with PD-L1 expression on macrophages. Allostimulatory capacity of macrophages was impaired and inversely correlated with PD-L1 expression and PD-L1/CD86 ratio. Finally, to verify that the cell contact–dependent inhibition of proliferation was due to macrophage´s PD-L1, we set up a co-culture in the presence of blocking antibody to PD-L1 and to IL-10. We showed that only PD-L1 blocking restored T-cell proliferation. These findings suggested that the effect of inhibitory marker PD-L1 overwhelmed the effect of co-stimulatory markers and down-regulated macrophage-T activation in presence of Neisseria gonorrhoeae infection. This study has important implications for understanding the mechanisms of clearance versus long-term persistence of N. gonorroheae infection and might be applicable for the development of new therapeutic strategies. Acknowledgements Dirección Investigación Facultad Odontología Universidad de Chile. Keywords: Neisseria gonorrhoeae, Macrophages, PD-L1, alloprolifertion, Immune Evasion Conference: IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología, Medellin, Colombia, 13 Oct - 16 Oct, 2015. Presentation Type: Oral Presentation Topic: Infectious and parasitic diseases Citation: Escobar A, Ortiz C, Garcia K, Rodas P and Acuña-Castillo C (2015). Increased PD-L1 expression and PD-L1/CD86 ratio on macrophages infected with Neisseria gonorrhoeae contributes to the functional Suppression of T-cell responses.. Front. Immunol. Conference Abstract: IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología. doi: 10.3389/conf.fimmu.2015.05.00303 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 15 Apr 2015; Published Online: 15 Sep 2015. * Correspondence: Dr. Alejandro Escobar, Universidad de Chile, Instituto de Investigación en Ciencias Odontológicas, Santiago, Chile, janodvm@gmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Alejandro Escobar Carolina Ortiz Killen Garcia Paula Rodas Claudio Acuña-Castillo Google Alejandro Escobar Carolina Ortiz Killen Garcia Paula Rodas Claudio Acuña-Castillo Google Scholar Alejandro Escobar Carolina Ortiz Killen Garcia Paula Rodas Claudio Acuña-Castillo PubMed Alejandro Escobar Carolina Ortiz Killen Garcia Paula Rodas Claudio Acuña-Castillo Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Tryptophan concentration is the main mediator of the capacity of adipose mesenchymal stromal cells to inhibit T-lymphocyte proliferation in vitro

Background aimsMesenchymal stromal cells (MSCs) have immunomodulatory properties that are mediated by cell-to-cell interactions and paracrine effects through soluble factors, among which tryptophan (Trp) conversion into kynurenine (Kyn) through the enzymatic activity of indoleamine 2,3-dioxygenase has been proven to be of special relevance. However, the respective role of Trp depletion and/or Kyn accumulation on the inhibition of T-cell proliferation by MSCs remains unclear. MethodsThe effect of supplementation with increasing concentrations of Trp on the capacity of MSCs to inhibit T-lymphocyte proliferation in vitro was investigated. ResultsWe report that Trp supplementation impairs the capacity of adipose mesenchymal stromal cells (ASCs) to inhibit T-cell proliferation, despite the accumulation of very high concentrations of Kyn in the medium (>200 μmol/L). Moreover, Trp supplementation after 72 h of peripheral blood mononuclear cell:ASC co-culture, once the inhibitory effect of ASCs was established, reverted ASC inhibition and restored T-cell proliferation. Addition to stimulated lymphocytes of Kyn inhibited T proliferation in 3 of 10 peripheral blood mononuclear cell donors, but at different concentrations, suggesting that sensitivity of lymphocytes to Kyn might be donor-dependent. ConclusionsOur results confirm the relevance of Trp metabolism as a key mediator of the immunomodulatory properties of ASCs and clarify the respective roles of the Trp/Kyn balance.