Inhibit T-cell Function Research Articles

Salinomycin promotes T-cell proliferation by inhibiting the expression and enzymatic activity of immunosuppressive indoleamine-2,3-dioxygenase in human breast cancer cells

Indoleamine 2,3 dioxygenase (IDO) is upregulated in many tumor types, including breast cancer, and plays a reputable role in promoting tumor immune tolerance. The importance of the immunosuppressive mechanism of IDO by suppressing T-cell function has garnered profound interest in the development of clinical IDO inhibitors. Herein, we established a screening method with cervical HeLa cells to induce IDO expression using interferon-γ (IFN-γ). After screening our chemical library, we found that salinomycin potently inhibited IFN-γ-stimulated kynurenine synthesis with IC50 values of 3.36–4.66 μM in both human cervical and breast cancer cells. Salinomycin lowered the IDO1 and IDO2 expression with no impact on the expression of tryptophan-2,3-dioxygenase. Interestingly, salinomycin potently repressed the IDO1 enzymatic activity by directly targeting the proteins in cells. Molecular docking revealed an alignment that favors nucleophilic attack of salinomycin in the catalytic domain of IDO1. Activation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway by IFN-γ was significantly suppressed by salinomycin, via inhibiting the Jak1, Jak2, and STAT1/3 phosphorylation. Moreover, it inhibited IFN-γ-induced activation of the nuclear factor (NF)-κB pathway by inhibiting IκB degradation and NF-κB phosphorylation without affecting BIN1 expression. Furthermore, salinomycin significantly restored the proliferation of T cells co-cultured with IFN-γ-treated breast cancer cells and potentiated antitumor activity of cisplatin in vivo. These findings suggest that salinomycin suppresses kynurenine synthesis by inhibiting the catalytic activity of IDO1 and its expression by inhibiting the JAK/STAT and NF-κB pathways. Salinomycin warrants further investigation as a novel dual-functional IDO inhibitor for cancer immunotherapy.

Synergistic effects of low‑dose chemotherapy and T cells in renal cell carcinoma.

Renal cell carcinoma (RCC) is not sensitive to conventional radiotherapy and chemotherapy, and the effectiveness rate of molecular targeted therapy is low. Therefore, it is urgent to identify new treatment methods. Recently, adoptive T-cell therapy has provided a new option for cancer treatment. Furthermore, low-dose chemotherapy not only has no evident side effects and inhibitory effects on the human immune system, but can also enhance the immune activity of some effector cells. Therefore, it is surmised that the combination of different mechanisms of chemotherapy and immunotherapy could be a new treatment concept. In the present study, the effects of low-dose chemotherapy combined with T cells in the treatment of renal cell carcinoma were explored using cytotoxicity assays, enzyme-linked immunosorbent assay (ELISA), western blot analysis and flow cytometric analysis. The results revealed that low-dose chemotherapy and T cells had synergistic effects on tumor cell elimination in vitro. The transforming growth factor (TGF)-β signaling pathway may be involved in the inhibition of T-cell functions. The targeted inhibition of TGF-β signals may be a promising therapeutic strategy for the treatment of renal cancer. The present results provided a novel strategy for the combination of low-dose chemotherapy and T cells to enhance the therapeutic efficacy of RCC treatment.

P-342 Margetuximab combined with anti-PD-1 (MGA012) or anti-PD-1/LAG-3 (MGD013) +/- chemotherapy in first-line therapy of advanced/metastatic HER2+ gastroesophageal junction or gastric cancer

Trastuzumab (T), a monoclonal antibody (mAb) targeting HER2, is the standard of care 1st-line therapy for advanced HER2+ GEJ/GC patients (pts). Margetuximab (M), an investigational Fc-engineered anti-HER2 mAb, targets the same HER2 epitope but with higher affinity for both 158V (high binding) and 158F (low binding) alleles of activating Fc receptor CD16A. Data suggests M coordinately enhances both innate and adaptive immunity, including antigen-specific T-cell responses to HER2. PD-1 and LAG-3 are T-cell checkpoint molecules that suppress T-cell function.

PD-L1 expression by dendritic cells is a key regulator of T-cell immunity in cancer.

Inhibiting the programmed death-1 (PD-1) pathway is one of the most effective approaches to cancer immunotherapy, but its mechanistic basis remains incompletely understood. Binding of PD-1 to its ligand PD-L1 suppresses T-cell function in part by inhibiting CD28 signaling. Tumor cells and infiltrating myeloid cells can express PD-L1, with myeloid cells being of particular interest as they also express B7-1, a ligand for CD28 and PD-L1. Here we demonstrate that dendritic cells (DCs) represent a critical source of PD-L1, despite being vastly outnumbered by PD-L1+ macrophages. Deletion of PD-L1 in DCs, but not macrophages, greatly restricted tumor growth and led to enhanced antitumor CD8+ T-cell responses. Our data identify a unique role for DCs in the PD-L1-PD-1 regulatory axis and have implications for understanding the therapeutic mechanism of checkpoint blockade, which has long been assumed to reflect the reversal of T-cell exhaustion induced by PD-L1+ tumor cells.

Abstract A16: Natural killer cell therapy efficacy is enhanced through inhibition of myeloid-derived suppressor cell trafficking

Abstract Immunotherapies designed to activate or replace T-cell effector immunity, such as immune checkpoint blockade or adoptive transfer of T cells, are limited by the ability of subpopulations of tumor cells to escape T-cell immunity. This can occur through immunologic selection of tumor cells that present weak T-cell antigens or harbor genomic or epigenetic alterations that alter antigen processing or presentation or responses to interferon. Immunotherapy designed to activate or replace NK cell immunity may overcome these limitations through antigen and MHC class I-independent tumor cell recognition and killing. However, similar to suppression of T-cell function, local immunosuppression within the tumor microenvironment may limit the function of NK cells. We hypothesized that immature, immunosuppressive myeloid cells (myeloid derived suppressor cells, or MDSCs) may limit optimal function of NK cells and that NK cell control of tumors can be enhanced through abrogation of MDSC trafficking and tumor infiltration. To model new NK cell therapy products that are available for clinical investigation, we studied how MDSCs alter the ability of culturable murine NK (KIL) cell line to kill oral cancer cells. We used an oral cancer model, MOC2, that has few genomic alterations and forms highly aggressive, non-T cell-inflamed tumors in wild-type C57BL/6 mice. CXCR2+ neutrophilic-MDSCs (PMN-MDSCs) sorted from MOC2 tumors significantly suppressed the ability of KIL to kill MOC2 tumor cells in vitro as assessed by real-time impedance analysis. NK suppression mediated by PMN-MDSC appeared to be mediated by both cell surface TGFβ and arginase. In vivo, we aimed to block chemotaxis and MOC2 tumor infiltration of CXCR2+ PMN-MDSC with the use of SX-682, a clinical-stage small-molecule inhibitor of CXCR1/2. After validating that SX-682 blocks tumor PMN-MDSC trafficking into tumors, we treated mice bearing established MOC2 tumors with SX-682 and adoptive transfer of KIL, alone or in combination. While SX-682 alone produced no tumor growth inhibition (TGI) and KIL adoptive transfer alone produced modest TGI, the combination resulted in significant TGI and prolonged survival of mice. Immune correlative analysis revealed increased tumor infiltration of adoptively transferred KIL with inhibition of PMN-MDSC tumor infiltration. These data suggest that the efficacy of NK cell therapies can be enhanced with PMN-MDSC inhibition and support the clinical investigation of chemokine receptor inhibitors such as SX-682 in combination with NK cell therapy products. Citation Format: Sarah Greene, Yvette Robbins, Paul E. Clavijo, Dean Maeda, John Zebala, Jeffrey Schlom, Claudia Palena, Clint Allen. Natural killer cell therapy efficacy is enhanced through inhibition of myeloid-derived suppressor cell trafficking [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr A16.

Development and functional analysis of an anticancer T-cell medicine with immune checkpoint inhibitory ability.

Adoptive cell therapy using patients' own T-cells is expected to be an ideal cancer treatment strategy with excellent antitumor effects and low side effects. However, this therapy targeting solid tumors is unlikely to be effective because tumor tissues have an environment that suppresses T-cell function. In particular, interaction between programmed death-1 (PD-1) and its ligand (PD-L1) inhibits T-cell activation by which T-cells eliminate tumor cells. Here, we attempted to develop T-cells that can exert potent antitumor activity even in tumor tissues by genetically modifying them to express the anti-PD-L1 membrane-anchoring type single chain variable fragment (M-scFv) that can inhibit PD-L1/PD-1 interaction. Anti-PD-L1 M-scFv could be expressed on T-cells while maintaining PD-L1-binding ability. Although T-cell proliferation induced by CD3 stimulation was decreased depending on the PD-L1 stimulation intensity, M-scFv-expressing T-cells showed high proliferative activity even in the presence of PD-L1 by avoiding the PD-L1/PD-1-mediated suppression. Furthermore, M-scFv-expressing T-cells showed higher cytotoxic activity against PD-L1high tumor cells than that of mock T-cells. The effect of PD-L1/PD-1 blockade was more pronounced when the therapeutic target was low-antigenic tumor cells with low major histocompatibility complex expression, presenting only the shared antigen. These results indicated that anti-PD-L1 M-scFv expression was functional in avoiding T-cell dysfunction by PD-L1/PD-1 interaction. Our concept of anti-PD-L1 M-scFv-expressing T-cells is thus expected to improve the efficacy of T-cell therapy and contribute to simplify the treatment system and reduce treatment costs compared with the combination therapy of T-cells and antibodies.

Abstract A94: Identification of potent, insurmountable A2a antagonists for modulation of the tumor microenvironment

Abstract Adenosine is elevated in the tumor microenvironment and plays a critical role in suppressing T-cell function through high-affinity interaction with the A2a receptor. Genetic deficiency of A2a in mice is associated with enhanced cytotoxic responses and reduced tumor burden in syngeneic models. These effects are mimicked by small-molecule A2a antagonists and some of these compounds are currently being evaluated in clinical trials for the treatment of solid tumors, particularly in combination with checkpoint inhibitors. However, high levels of adenosine in the tumor microenvironment may pose a challenge for the development of A2a antagonists unless they possess the appropriate pharmaceutical profile. A series of potent antagonists were identified that displayed sub-nanomolar binding activity and selectivity over other adenosine receptors. Within this series, compounds were identified that maintained activity across a broad range of adenosine concentrations. These compounds behaved as insurmountable antagonists in a functional assay utilizing CHO cells expressing human recombinant A2a and displayed slow dissociation kinetics in a FRET-based receptor-binding assay. This series is exemplified by ARX1598, which had a Ki of 0.08nM in the A2a receptor-binding assay and an estimated KB of 0.06 nM in the recombinant cell-based assay. Schild analysis revealed that this compound behaved as an insurmountable antagonist and had a markedly slower off-rate than comparator competitive antagonists. The adenosine receptor agonist NECA suppressed cytokine production by peripheral blood mononuclear cells activated by anti-CD3/anti-CD28. ARX1598 prevented NECA-mediated suppression of cytokine production by human peripheral blood mononuclear cells (EC50= 6 nM) and prevented elevation of pCREB induced by high concentrations of NECA (3μM) in human whole blood (IC50=22nM). The potency of ARX1598 in the human whole blood assay was 50-100 times higher than comparator compounds currently being evaluated in cancer trials. These data illustrate that it is possible to identify potent compounds that have the potential to modulate the adenosine pathway in the tumor microenvironment. Citation Format: Roy Pettipher, Jonathan White, Viral Patel, Ben Moulton, Soraya Pores, Marta Assuncao, Karolina Gherbi, K. Sengmany, Elisabeth Rosethorne, Peter Finan, Steven Charlton, Clive McCarthy. Identification of potent, insurmountable A2a antagonists for modulation of the tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A94.

Abstract B13: Immune cells, tumor-initiating cells, and drug sensitivity in claudin-low TNBC: A delicate balance

Abstract Background: Breast cancer is a heterogeneous disease. Based on gene expression profiling, it can be classified into at least 5 subtypes. They are luminal A and B, normal breast-like, Her2-positive, basal-like, and newly described claudin-low. Among all these subtypes, basal-like and claudin-low tumors have relatively poor prognosis. Claudin-low tumors are characterized by the low expression of cell-cell junction proteins like claudin 3 and E-cadherin along with an enrichment of EMT markers such as SNAI1 and SNAI2 and high levels of mesenchymal markers such as vimentin. Claudin-low tumors are generally more aggressive and the rate of response to primary chemotherapy is around 32%. Initial data have shown that p-53 null claudin-low tumors are sensitive to cyclophosphamide treatment. Cyclophosphamide (CTX) is a chemotherapy drug that has been used in lymphoma, leukemia and some solid tumors. At low and continuous doses, it shows immunostimulatory properties. The overall goal of this proposal is to test the therapeutic response of claudin-low tumors to cyclophosphamide treatment and define the functional role of the immune system in a mouse model of claudin-low breast cancer. Preliminary data have shown that tumor cells in a p53 null claudin-low murine model (T12) possess tumor-initiating abilities and have STAT3 activation. Initial data in claudin-low T12 tumors also suggest that CTX induces CD4+ and CD8+ T-cell homing to the primary tumor. Additionally, wild-type Balb/c mice implanted with T12 tumors respond favorably to CTX treatment as compared to T cell-deficient NSG mice. Our studies have shown that treatment-resistant claudin-low tumors have a high tumor-associated macrophage (TAM) infiltrate. Tumors with poor response to CTX show higher levels of vimentin staining as compared to responsive tumors. Depletion of specific T-cell subsets to determine their functional importance in the response to CTX shows that while CD8+ T-cell depletion does not have a significant effect on treatment response, depletion of CD4+ T cells results in impaired tumor response to CTX treatment. Additionally, TAMs increase upon CD4+ T-cell depletion. We hypothesize that CTX treatment can sensitize T12 primary tumors to CD4+ T-cell mediated apoptosis. We further hypothesize that TAMS can suppress T-cell function and this, along with changes in the tumor EMT status, can promote chemoresistance. TAMs can also interact with tumor-initiating cells (TIC) within claudin-low tumors via the STAT3 pathway to promote survival and proliferation of chemotherapy resistant TICs. Furthermore, depleting immunosuppressive cells such as TAMs may provide us with an additional therapeutic modality for chemotherapy-resistant breast cancer in combination with checkpoint immunotherapy. Reference: 1. Prat A et al. Phenotypic and molecular characterization of the claudin-low intrinsic subtype of breast cancer. Breast Cancer Res 2010;12:R68. Citation Format: Swarnima Singh, Se-Jin Kim, Xiang Zhang, Jeffrey Rosen. Immune cells, tumor-initiating cells, and drug sensitivity in claudin-low TNBC: A delicate balance [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B13.

Programmed Cell Death-1: Programmed Cell Death-Ligand 1 Interaction Protects Human Cardiomyocytes Against T-Cell Mediated Inflammation and Apoptosis Response In Vitro

Aim: Immunological checkpoint therapy is considered a powerful method for cancer therapy and acts by re-activating autologous T cells to kill the cancer cell. Myocarditis cases have been reported in cancer patients after immunological therapy; for example, nivolumab treatment is a monoclonal antibody that blocks programmed cell death-1/programmed cell death ligand-1 ligand interaction. This project provided insight into the inflammatory response as a benchmark to investigate the potential cardiotoxic effect of T cell response to the programmed cell death-1 (PD-1)/programmed cell death ligand-1 (PD-L1) axis in regulating cardiomyocyte injury in vitro. Methods and Results: We investigated cardiomyopathy resulted from the PD-1/PD-L1 axis blockade using the anti-PD-1 antibody in Rockefeller University embryonic stem cells-derived cardiomyocytes (RUES2-CMs) and a melanoma tumor-bearing murine model. We found that nivolumab alone did not induce inflammatory-related proteins, including PD-L1 expression, and did not induce apoptosis, which was contrary to doxorubicin, a cardiotoxic chemotherapy drug. However, nivolumab was able to exacerbate the immune response by increasing cytokine and inflammatory gene expression in RUES2-CMs when co-cultured with CD4+ T lymphocytes and induced apoptosis. This effect was not observed when RUES2-CMs were co-cultured with CD8+ T lymphocytes. The in vivo model showed that the heart function of tumor-bearing mice was decreased after treatment with anti-PD-1 antibody and demonstrated a dilated left ventricle histological examination. The dilated left ventricle was associated with an infiltration of CD4+ and CD8+ T lymphocytes into the myocardium. PD-L1 and inflammatory-associated gene expression were significantly increased in anti-PD-1-treated tumor-bearing mice. Cleaved caspase-3 and mouse plasma cardiac troponin I expressions were increased significantly. Conclusion: PD-L1 expression on cardiomyocytes suppressed T-cell function. Blockade of PD-1 by nivolumab enhanced cardiomyocyte inflammation and apoptosis through the enhancement of T-cell response towards cardiomyocytes.

Abstract B54: Single-cell analytics in claudin-low TNBC reveals immunostimulatory effects of low-dose metronomic cyclophosphamide treatment

Abstract Breast cancer is a heterogeneous disease. It can be classified into at least five subtypes: luminal A and B, normal breast like, Her2 positive, basal like, and claudin low. Among these, claudin-low tumors have relatively poor prognosis. These tumors are characterized by the low expression of cell-cell junction proteins along with an enrichment of EMT markers. They are more aggressive and the rate of response to primary chemotherapy is around 32%. Initial data have shown that murine p53 null claudin-low tumors are only sensitive to cyclophosphamide treatment. Cyclophosphamide (CTX) is a chemotherapy drug that has been used in lymphoma, leukemia, and some solid tumors. At low and continuous doses, it shows immunostimulatory properties. In this study we describe the implementation of a novel combination treatment for claudin-low TNBC as informed by combined scRNA-seq and V(D)J seq. Immune profiles of a murine claudin-low tumor model (T12) shows heavy infiltration by tumor-associated macrophages (TAMs) pretreatment. Initial data in T12 tumors also suggest that CTX induces CD4+ and CD8+ T-cell homing to the primary tumor. Wild-type Balb/c mice implanted with T12 tumors respond favorably to CTX treatment as compared to T cell-deficient NSG mice. We combined 5´ scRNA-seq with V(D)J seq to gain an insight into how immune cell subpopulations change during the course of treatment. Combining tSNE plots for untreated and treated tumors revealed the emergence of new transcriptionally distinct populations in the CTX treated group, which included six T cell subpopulations. Projection of T cells along pseudotime using Monocle3 revealed a shift from naive CD8+ and CD4+ T cells to a terminal effector CD8+ T-cell phenotype and CD4+ effector memory cells in treated tumors while untreated tumors have higher numbers of exhausted Tigit+ CD8+ T cells and Tregs. V(D)J analysis revealed higher numbers of unique TCRs in CTX-treated tumors and greater clonal expansion of T cells. However, we noted a persistence of TAMs in our model even after treatment as well as high recurrence rates upon cessation of treatment. We then examined markers specific to the TAM subsets in the treated and untreated tumors and observed that TAMs expressed high levels of Csf1r. A chow containing PLX3397(iCSF1R) was employed to inhibit CSF1R, a macrophage differentiation/recruitment factor. Combination treatment with CTX and the iCSF1R chow lead to long-term regression of T12 tumors. Rechallenging these mice resulted in much slower tumor growth, indicating the formation of T cell memory. scRNA-seq and V(D)J seq is being performed on these samples to identify the clonal dynamics of effector T cells that assist in this striking regression. We hypothesize that CTX treatment can sensitize T12 primary tumors to T cell-mediated apoptosis, and that TAMS can suppress T-cell function and promote chemoresistance. Furthermore, depleting these TAMs in combination with CTX treatment may lead to long-term durable regression of both the primary tumor and metastases. Citation Format: Swarnima Singh, Ying-Wooi Wan, Nigel Lee, Se-Jin Kim, Zhandong Liu, Charles M. Perou, Xiang Zhang, Jeffrey Rosen. Single-cell analytics in claudin-low TNBC reveals immunostimulatory effects of low-dose metronomic cyclophosphamide treatment [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B54.

Abstract A97: Neutrophil extracellular traps (NETs) promote immune escape and metastatic growth after surgical stress

Abstract Introduction: Surgery induces local and systemic inflammatory and immunosuppressive disturbances that correlate with adverse cancer outcomes. Following surgical stress, NETs promote the growth of liver micrometastases. We hypothesize that NETs support metastatic growth by promoting immune escape. Methods: Liver ischemia reperfusion was performed 5 days after a murine liver metastatic model was established. Results: Liver PD-L1 expression increased and peaked between 6-48 hours after reperfusion. A smaller peak of PD-L1 was observed after 2 weeks as the metastatic tumors grew. During the first 48 hours, the predominant source of PD-L1 was CD45+-CD11b+-Ly6G+ cells. PD-L1 expression was significantly decreased when NETs were inhibited either genetically by using PAD4KO mice or DNAse injections. In vitro, we confirmed both human and murine NETs express high levels of PD-L1. After liver I/R, tumor-infiltrating T cells expressed inhibitory receptors and were highly dysfunctional with reduced cytokine production and proliferative capacity in the NET-rich tumors compared to tumors from sham mice. T-cell exhaustion was reversed in mice treated with DNAse or PAD4KO mice. Similar results were observed in vitro as NETs suppressed T-cell function via PDL-1/PD-1 interaction. Treating mice with anti-PD-L1 during the first 48 hours after reperfusion reversed T-cell exhaustion and decreased tumor growth. Conclusion: NETs induced by surgical stress cause immune escape by secreting PD-L1, increasing T-cell exhaustion, and upregulation of immunosuppressive ligands on tumor cells. These findings suggest avenues for overcoming the perioperative accelerated tumor growth and immunosuppressive state and ultimately improving long-term oncologic outcomes. Citation Format: Hamza O. Yazdani, Christof T. Kaltenmeier, David Geller, Samer Tohme. Neutrophil extracellular traps (NETs) promote immune escape and metastatic growth after surgical stress [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A97.

Abstract B101: Molecular characteristics of tumor-associated macrophages in human melanoma metastases

Abstract Tumor-associated macrophages (TAMs) have been implicated in suppressing T-cell activity against cancer cells, representing an important target for the development of new immune therapy combinations. Accumulating evidence indicates that increased numbers of TAMs infiltrating cancer including melanoma are associated with poor prognosis. However, TAMs remain poorly defined in human cancers as they can exhibit a range of different molecular phenotypes and functional properties, and their cellular precursors are also uncertain, necessitating further investigation to enable their therapeutic manipulation. Using multiplex immunohistochemistry up to 7 colors and flow cytometry, here we sought to examine molecular characteristics of TAMs in human melanoma metastases to gain better understanding of the mechanisms driving their immune-suppressive activity in tumors as wells as the pathways recruiting their precursors. In lymph node and dermal melanoma metastases, we found that a population of TAMs expressing the marker CD163 frequently express molecules that are highly suppressive of T-cell function, including the PD-1 ligands PD-L1 and PD-L2, IDO (indoleamine 2,3-dioxygenase), TGF-β, and IL-10. In particular, the predominant source of PD-L1 is not the melanoma cells present but the infiltrating CD163+ TAMs. We also observed that many of the CD163+ TAMs downmodulate MHC class II molecules, represented by HLA-DR, suggesting they have suppressed capacity to present melanoma cell antigens to T cells. CD163+ TAMs in melanoma metastases often express weak levels of CD14 and CCR2, while cells adjacent to them express these markers brightly, suggesting that these TAMs are likely to derive from recently extravasated monocytes. Further analysis confirmed that CCL2, the chemokine ligand for CCR2, is indeed present in many melanoma tissue samples examined. Interestingly, the majority of CD163+CD14+ TAMs in melanoma metastases also express high levels of CD16, suggesting that these TAMs are either more closely related to the nonclassical CD14+CD16+ monocytes or derive from the classical CD14+CD16- monocytes that subsequently gain CD16 expression after entering the tumor tissue. In vitro, we were able to generate cells from primary human monocytes that recapitulate the phenotype of CD163+ TAMs observed in situ and are capable of suppressing T cells, which will provide us a valuable tool to carry out functional studies to reveal how TAMs suppress T-cell function and how to reverse such activities. Collectively, our data demonstrate the complex molecular features of CD163+ TAMs in melanoma metastases, necessitating further investigations to determine the dominant T cell-suppressive mechanisms used by TAMs and to prioritize therapeutic targets. Citation Format: Saem Park, Anna Brooks, Chun-Jen Chen, Rod Dunbar. Molecular characteristics of tumor-associated macrophages in human melanoma metastases [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B101.

Short-term prognostic effects of circulating regulatory T-Cell suppressive function and vascular endothelial growth factor level in patients with non-small cell lung cancer and obstructive sleep apnea

ObjectiveTo determine if suppressive function of regulatory T-cells (Tregs) and vascular endothelial cell growth factor (VEGF) levels are closely associated with prognosis of patients with non-small cell lung cancer (NSCLC) and obstructive sleep apnea (OSA). MethodsPeripheral blood from 20 OSA patients, 44 newly diagnosed NSCLC patients with (n = 22) and without (n = 22) OSA was collected. Forkhead box protien 3 plus (Foxp3+) and CTLA-4+ Tregs ratio were analyzed with flow cytometry. Levels of VEGF, IL-10 and TGF-β1 were analyzed with enzyme-linked immuno sorbent assay. NSCLC patients with and without OSA were followed up for two years. Optimal cutoff values were determined by receiver operating characteristic curves. Survival analysis were performed using the Kaplan–Meier test. ResultsNSCLC patients with OSA showed higher Foxp3+Tregs ratio, higher plasma VEGF and TGF-β1 levels when compared with NSCLC patients without OSA (P < 0.05). In NSCLC patients with OSA or not, subjects with higher Foxp3+Treg ratio, higher TGF-β1 and VEGF levels tended to have poor mean survival time and two-year overall survival (OS, Foxp3+Treg: 636.7 vs. 704.8 days, 59.0% vs. 82.6%, P = 0.125; TGF-β1: 637.8 vs. 698.4 days, 57.0% vs. 84.4%, P = 0.054; VEGF: 642.9 vs. 677.5 days, 48.6% vs. 81.3%, P = 0.074). Multivariate Cox regression adjusted for disease stage and receipt of systemic treatments, confirmed the links between high VEGF level and worse OS (HR: 1.003; 95% CI: 1.001–1.005; P = 0.021). ConclusionsOSA may up-regulate the expression of circulating TGF-β1, VEGF and Foxp3+Tregs expression in NSCLC patients. Elevated VEGF level is closely associated with worse short-term survival in NSCLC patients with OSA or not.

Margetuximab (M) combined with anti-PD-1 (MGA012) or anti-PD-1/LAG-3 (MGD013) +/- chemotherapy (CTX) in first-line therapy of advanced/metastatic HER2+ gastroesophageal junction (GEJ) or gastric cancer (GC).

TPS468 Background: Trastuzumab (T), a monoclonal antibody (mAb) targeting HER2, is standard of care palliative 1st-line therapy for advanced HER2+ GEJ/GC patients (pts). M, an Fc-engineered anti-HER2 mAb, targets the same HER2 epitope but with higher affinity for both 158V (high binding) and 158F (low binding) alleles of activating Fc receptor CD16A. M coordinately enhanced both innate and adaptive immunity, including antigen-specific T-cell responses to HER2. PD-1 and LAG-3 are T-cell checkpoint molecules that suppress T-cell function. MGA012 (INCMGA00012) is a humanized, hinge-stabilized, IgG4 κ anti-PD-1 mAb blocking binding of PD-L1 or PD-L2 to PD-1. MGD013 is a humanized Fc-bearing bispecific tetravalent protein that binds to both PD-1 and LAG-3, inhibiting their respective ligand binding. We previously reported that a CTX-free regimen of M+PD-1 blockade was well tolerated in GEJ/GC pts, and induced a 30% objective response rate (ORR). This was 2- to 3-fold greater than in historical controls with checkpoint inhibitors alone. This registration-directed trial assesses efficacy, safety, and tolerability of M+checkpoint inhibition ± CTX in metastatic/locally advanced, treatment-naïve, HER2+ GEJ/GC pts. Methods: This is a 2-cohort, adaptive open-label phase 2/3 study. The first single arm, CTX-free cohort A evaluates M+MGA012 in HER2+ (immunohistochemistry [IHC] 3+) and PD-L1+ (excluding microsatellite instability high) pts. After 40 pts are evaluated for response/safety, 60 more pts will be enrolled if the threshold for continuation is met. In randomized cohort B, HER2+ (IHC 3+ or 2+/fluorescent in situ hybridization+) pts are enrolled irrespective of PD-L1 status. Part 1 randomizes pts to 1 of 4 arms (50 pts each): control arm (T+CTX) or 1 experimental arm (M+CTX; M+CTX+MGA012; M+CTX+MGD013). CTX is investigator’s choice XELOX or mFOLFOX-6. Part 2 consists of control (T+CTX) vs 1 experimental arm (M+CTX) + either MGA012 or MGD013, depending on results from part 1; with 250 pts each. The primary efficacy endpoint for cohort A (both parts) is ORR per RECIST 1.1; for cohort B part 2 it is overall survival.

Programmed cell death protein 1 (PD-1) in infection.

Exhausted and dysfunctional T cells triggered by infection and cancer render the immune system unable to eliminate these pathogens. Pharmacologic blockade of the surface receptors that inhibit T-cell function has shown remarkable success in patients with various malignancies. In this Review, we discuss the emerging evidence of inhibiting checkpoint pathways as a potential role in controlling or clearing infectious diseases. Though interesting tendencies, much work is still needed in order to develop safe strategies that can be translated into clinically relevant outcomes in patients with infections.

Yap suppresses T-cell function and infiltration in the tumor microenvironment.

A major challenge for cancer immunotherapy is sustaining T-cell activation and recruitment in immunosuppressive solid tumors. Here, we report that the levels of the Hippo pathway effector Yes-associated protein (Yap) are sharply induced upon the activation of cluster of differentiation 4 (CD4)-positive and cluster of differentiation 8 (CD8)-positive T cells and that Yap functions as an immunosuppressive factor and inhibitor of effector differentiation. Loss of Yap in T cells results in enhanced T-cell activation, differentiation, and function, which translates in vivo to an improved ability for T cells to infiltrate and repress tumors. Gene expression analyses of tumor-infiltrating T cells following Yap deletion implicates Yap as a mediator of global T-cell responses in the tumor microenvironment and as a negative regulator of T-cell tumor infiltration and patient survival in diverse human cancers. Collectively, our results indicate that Yap plays critical roles in T-cell biology and suggest that Yap inhibition improves T-cell responses in cancer.

Pembrolizumab-Induced Diabetes Mellitus Presenting as Diabetic Ketoacidosis in a Patient With Metastatic Colonic Adenocarcinoma

Immunotherapy drugs are gaining popularity in the treatment of certain malignancies due to the success of these agents in recent clinical trials. Pembrolizumab is an immune checkpoint inhibitor that acts via binding to programmed cell death 1 (PD-1) receptors on T-cells, allowing for the constitutive activation of T-cells to fight malignant tumor cells. Immune checkpoint molecules such as PD-1 act to inhibit T-cell function, promoting tolerance to self-antigens. Inhibition of these molecules may lead to increased T-cell activation against cancer cells, but also against healthy tissue, leading to the side effects of these medications known as immune-related adverse events. In this article, we present the case of a 77-year-old female with a history of metastatic colonic adenocarcinoma presenting with new-onset diabetes mellitus and diabetic ketoacidosis in the setting of receiving pembrolizumab chemotherapy. Our patient was treated with hydration, insulin therapy, and management of her electrolytes, ultimately being discharged with the need for home insulin therapy to manage her new-onset diabetes. There are no current guidelines for the management or surveillance of patients receiving pembrolizumab chemotherapy, and further research should be done to determine which patients are at highest risk to developing this rare but potentially lethal side effect.

Discovery of Small Molecules for the Reversal of T Cell Exhaustion

Inhibitory receptors (IRs) function as critical regulators of immune responses by tempering Tcell activity. In humans, several persisting viruses as well as cancers exploit IR signaling by upregulating IR ligands, resulting in suppression of Tcell function (i.e., exhaustion). This allows escape from immune surveillance and continuation of disease. Here, we report the design, implementation, and results of a phenotypic high-throughput screen for molecules that modulate CD8+ Tcell activity. We identify 19 compounds from the ReFRAME drug-repurposing collection that restore cytokine production and enhance the proliferation of exhausted Tcells. Analysis of our top hit, ingenol mebutate, a protein kinase C (PKC) inducing diterpene ester, reveals a role for this molecule in overriding the suppressive signaling cascade mediated by IR signaling on Tcells. Collectively, these results demonstrate a disease-relevant methodology for identifying modulators of Tcell function and reveal new targets for immunotherapy.

Mass Cytometry Identifies a Novel Signature for Myeloid-Derived Suppressor-Cells in Waldenstrom's Macroglobulinemia

Myeloid derived suppressor cells (MDSC) are a heterogeneous population of undifferentiated myeloid cells that are expanded and activated in pathological conditions and have the ability to potently suppress T-cell function and thereby contribute to immunosuppression and tumor progression. While there have been studies showing a role for MDSC in a variety of hematological malignancies, no data is available indicating that MDSCs contribute to the tumor progression in Waldenstrom's Macroglobulinemia (WM), an indolent lymphoma characterized by bone marrow (BM) infiltration of lymphoplasmacytic (LPL) cells and increased secretion of monoclonal IgM. In previous work, we have found increased GM-CSF and reduced arginine and cysteine in the BM microenvironment in WM. We hypothesized that this was due to the presence and activity of MDSCs in WM. BM aspirates from WM patients (n=17) were therefore processed to isolate LPL (CD19+/CD138+) cells from the rest of the BM cells (CD19-/CD138-). Sorted (CD19-/CD138-) cells from BM of patients with WM were studied with flow cytometry. Using a sequential gating strategy (lack of lineage markers, low levels of HLADR, CD33+, CD11b+) we identified a population of MDSCs that were then subdivided using CD14 and CD15 expression into total-, monocytic-, or granulocytic- MDSCs (m-MDSC, g-MDSC). We also analyzed unsorted BM cells using cytometry by time-of-flight (CyTOF) in order to further identify and phenotypically characterize the BM MDSC population in a group of WM patients with smoldering (asymptomatic) disease, symptomatic disease, or in remission post-treatment. BM samples from normal subjects were used as a control. Flow cytometry data showed significant higher numbers of MDSC subsets expressing PD-L1 and Arginase1 in WM patients when compared to the normal samples. BM cells from WM patients (n=18) then were compared to controls (n=11), and the absolute number of the total MDSC (p=0.05), m-MDSC (p=0.002), g-MDSC (p=0.02) was increased in WM specimens. When MDSCs from WM or normal monocytes from healthy controls were co-cultured with activated T-cells, the proliferation of activated T-cells in the presence of MDSCs from WM patients was impaired compared to controls, confirming the suppressive role of MDSCs. We then performed high dimensional analysis of the total BM MDSC cells using t-SNEand identified phenotypically distinct MDSC cell populations in the BM that were differentially present when healthy controls were compared to patients with smoldering WM or those with WM needing treatment. Specifically, WM patients needing treatment had increased numbers of a distinct MDSC population that was highly positive for CD163, and CD138. Moreover, conventional markers denoting m-MDSC and g-MDSC, such as CD14 and CD15, were highly expressed in all populations and their pattern of expression did not specifically define the MDSC subtypes, indicating that high dimensional phenotyping further details the MDSC sub-compartments beyond the conventional categorization of MDSC using conventional cytometry. In summary, we find that MDSCs are increased in the BM of WM patients compared to controls. MDSCs expressing CD163 and CD138 increase when WM patients become symptomatic and require therapy. Furthermore, MDSCs in the BM of WM patients suppress T-cell function and likely contribute to progression of the disease. MDSCs in the BM therefore present a therapeutic target that should be explored in WM patients. Disclosures Ansell: Bristol Myers Squibb: Other: research funding for clinical trials; Merck: Other: research funding for clinical trials; AI Therapeutics: Other: research funding for clinical trials; Affimed: Other: research funding for clinical trials; Takeda: Other: research funding for clinical trials; Pfizer: Other: research funding for clinical trials; Regeneron: Other: research funding for clinical trials; Seattle Genetics: Other: research funding for clinical trials.

Preclinical Development of the Anti-LAG-3 Antibody REGN3767: Characterization and Activity in Combination with the Anti-PD-1 Antibody Cemiplimab in Human PD-1xLAG-3-Knockin Mice.

In the tumor microenvironment, multiple inhibitory checkpoint receptors can suppress T-cell function, thereby enabling tumor immune evasion. Blockade of one of these checkpoint receptors, PD-1, with therapeutic antibodies has produced positive clinical responses in various cancers; however, the efficacy of this approach can be further improved. Simultaneously targeting multiple inhibitory checkpoint receptors has emerged as a promising therapeutic strategy. Here, we report the development and characterization of REGN3767, a fully human IgG4 antibody targeting LAG-3, another inhibitory receptor on T cells. REGN3767 binds human and monkey LAG-3 with high affinity and specificity and blocks the interaction of LAG-3 with its ligand, MHC class II. In an engineered T-cell/antigen-presenting cell bioassay, REGN3767 alone, or in combination with cemiplimab (REGN2810, human anti-PD-1 antibody), blocked inhibitory signaling to T cells mediated by hLAG-3/MHCII in the presence of PD-1/PD-L1. To test the in vivo activity of REGN3767 alone or in combination with cemiplimab, we generated human PD-1xLAG-3 knockin mice, in which the extracellular domains of mouse Pdcd1 and Lag3 were replaced with their human counterparts. In these humanized mice, treatment with cemiplimab and REGN3767 showed increased efficacy in a mouse tumor model and enhanced the secretion of proinflammatory cytokines by tumor-specific T cells. The favorable pharmacokinetics and toxicology of REGN3767 in nonhuman primates, together with enhancement of antitumor efficacy of anti-PD-1 antibody in preclinical tumor models, support its clinical development.