Programmed Cell Death-1 Blockade Research Articles

Targeting immune checkpoints in lymphoma.

The purpose of this article is to discuss the tumor microenvironment in lymphoma, and to review potential immune targets that are now becoming relevant because of clinical responses seen with the use of immune checkpoint inhibitors. Recent data have shown that cells within the immune microenvironment in lymphoma express programmed death ligand-1 (PD-L1) and many of the intratumoral T cells with an exhausted immune phenotype express programmed cell death-1 (PD-1). This provides a novel opportunity to inhibit the immune checkpoints and initial clinical trials utilizing antibodies that block the interaction between PD-1 and PD-L1 have demonstrated significant clinical responses in various lymphomas, including Hodgkin lymphoma. The use of immune checkpoint inhibitors, including nivolumab and pembrolizumab, in relapsed and refractory patients with lymphoma is proving highly successful. Patients with Hodgkin lymphoma, in particular, have a very high response rate to PD-1 blockade and responses in these patients appear durable.

Assays for predicting and monitoring responses to lung cancer immunotherapy.

Immunotherapy has become a key strategy for cancer treatment, and two immune checkpoints, namely, programmed cell death 1 (PD-1) and its ligand (PD-L1), have recently emerged as important targets. The interaction blockade of PD-1 and PD-L1 demonstrated promising activity and antitumor efficacy in early phase clinical trials for advanced solid tumors such as non-small cell lung cancer (NSCLC). Many cell types in multiple tissues express PD-L1 as well as several tumor types, thereby suggesting that the ligand may play important roles in inhibiting immune responses throughout the body. Therefore, PD-L1 is a critical immunomodulating component within the lung microenvironment, but the correlation between PD-L1 expression and prognosis is controversial. More evidence is required to support the use of PD-L1 as a potential predictive biomarker. Clinical trials have measured PD-L1 in tumor tissues by immunohistochemistry (IHC) with different antibodies, but the assessment of PD-L1 is not yet standardized. Some commercial antibodies lack specificity and their reproducibility has not been fully evaluated. Further studies are required to clarify the optimal IHC assay as well as to predict and monitor the immune responses of the PD-1/PD-L1 pathway.

PD-1 and Tim-3 pathways are associated with regulatory CD8+ T-cell function in decidua and maintenance of normal pregnancy

CD8+ T cells are critical in the balance between fetal tolerance and antiviral immunity. T-cell immunoglobulin mucin-3 (Tim-3) and programmed cell death-1 (PD-1) are important negative immune regulatory molecules involved in viral persistence and tumor metastasis. Here, we demonstrate that Tim-3+PD-1+CD8+ T cells from decidua greatly outnumbered those from peripheral blood during human early pregnancy. Co-culture of trophoblasts with CD8+ T cells upregulated PD-1+ and/or Tim-3+ immune cells. Furthermore, the population of CD8+ T cells co-expressing PD-1 and Tim-3 was enriched within the intermediate memory subset in decidua. This population exhibited high proliferative activity and Th2-type cytokine producing capacity. Blockade of Tim-3 and PD-1 resulted in decreased in vitro proliferation and Th2-type cytokine production while increased trophoblast killing and IFN-γ producing capacities of CD8+ T cells. Pregnant CBA/J females challenged with Tim-3 and/or PD-1 blocking antibodies were more susceptible to fetal loss, which was associated with CD8+ T-cell dysfunction. Importantly, the number and function of Tim-3+PD-1+CD8+ T cells in decidua were significantly impaired in miscarriage. These findings underline the important roles of Tim-3 and PD-1 pathways in regulating decidual CD8+ T-cell function and maintaining normal pregnancy.

Prostaglandin E2 and programmed cell death 1 signaling coordinately impair CTL function and survival during chronic viral infection.

More than 10% of the world’s population is chronically infected with HIV, HCV or HBV, which cause severe disease and death. These viruses persist in part because continuous antigenic stimulation causes deterioration of virus-specific cytotoxic T lymphocyte (CTL) function and survival. Additionally, antiviral CTLs autonomously suppress their responses to limit immunopathology by upregulating inhibitory receptors such as Programmed cell death 1 (PD-1). Identification and blockade of the pathways that induce CTL dysfunction may facilitate clearance of chronic viral infections. We have identified that the prostaglandin E2 (PGE2) receptors EP2 and EP4 are upregulated on virus-specific CTLs during chronic LCMV infection and suppress CTL survival and function. We showed that the combined blockade of PGE2 and PD-1 signaling was therapeutic in terms of improving viral control and augmenting the numbers of functional virus-specific CTLs. Thus, PGE2 inhibition is both independent candidate therapeutic target and a promising adjunct therapy to PD-1 blockade for treatment of HIV and other chronic viral infections.

Cancer immunology. Mutational landscape determines sensitivity to PD-1 blockade in non-small cell lung cancer.

Immune checkpoint inhibitors, which unleash a patient's own T cells to kill tumors, are revolutionizing cancer treatment. To unravel the genomic determinants of response to this therapy, we used whole-exome sequencing of non-small cell lung cancers treated with pembrolizumab, an antibody targeting programmed cell death-1 (PD-1). In two independent cohorts, higher nonsynonymous mutation burden in tumors was associated with improved objective response, durable clinical benefit, and progression-free survival. Efficacy also correlated with the molecular smoking signature, higher neoantigen burden, and DNA repair pathway mutations; each factor was also associated with mutation burden. In one responder, neoantigen-specific CD8+ T cell responses paralleled tumor regression, suggesting that anti-PD-1 therapy enhances neoantigen-specific T cell reactivity. Our results suggest that the genomic landscape of lung cancers shapes response to anti-PD-1 therapy.

T-cell exhaustion in chronic hepatitis B infection: current knowledge and clinical significance.

Hepatitis B virus (HBV) infection is the major cause of inflammatory liver disease, of which the clinical recovery and effective anti-viral therapy is associated with the sustained viral control of effector T cells. In humans, chronic HBV infection often shows weak or absent virus-specific T-cell reactivity, which is described as the ‘exhaustion' state characterized by poor effector cytotoxic activity, impaired cytokine production and sustained expression of multiple inhibitory receptors, such as programmed cell death-1 (PD-1), lymphocyte activation gene-3, cytotoxic T lymphocyte-associated antigen-4 and CD244. As both CD4+ and CD8+ T cells participate in the immune responses against chronic hepatitis virus through distinct manners, compelling evidences have been proposed, which restore the anti-viral function of these exhausted T cells by blocking those inhibitory receptors with its ligand and will pave the way for the development of more effective immunotherapeutic and prophylactic strategies for the treatment of chronic infectious diseases. A large number of studies have stated the essentiality of T-cell exhaustion in virus-infected diseases, such as LCMV, hepatitis C virus (HCV), human immunodeficiency virus infections and cancers. Besides, the functional restoration of HCV- and HIV-specific CD8+ T cells by PD-1 blockade has already been repeatedly verified, and also for the immunological control of tumors in humans, blocking the PD-1 pathway could be a major immunotherapeutic strategy. Although the specific molecular pathways of T-cell exhaustion remain ambiguous, several transcriptional pathways have been implicated in T-cell exhaustion recently; among them Blimp-1, T-bet and NFAT2 were able to regulate exhausted T cells during chronic viral infection, suggesting a distinct lineage fate for this sub-population of T cells. This paper summarizes the current literature relevant to T-cell exhaustion in patients with HBV-related chronic hepatitis, the options for identifying new potential therapeutic targets to treat HBV infection and highlights priorities for further study.

Covering the Cover

Covering the Cover

Programmed cell death-1 blockade in combination with stereotactic radiation in an orthotopic mouse model of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) develops in an environment of chronic inflammation. Curative treatments are plagued by frequent recurrences locally near the treated tumor, regionally within the inflamed liver, and systemically, and yet no adjuvant therapies are known to prevent recurrence. Here the efficacy of immunotherapy with stereotactic radiation (SBRT) in an immune-competent murine model is evaluated. A syngeneic HCC cell line (Hep 55.1c) is injected into the liver of C57BL/6 mice using ultrasound guidance. The tumors are measured and treated using computed tomography and a single dose of intravenous contrast that is taken up by resident liver macrophages allowing visualization of tumors for at least 30 days. Stereotactic radiation is delivered using a Small Animal Radiation Research Platform. Three doses of 250 ug anti-Programmed Cell Death-1 antibody (αPD-1) are delivered concurrently with 30 Gy SBRT in 3 fractions. This combination reduces the growth rate of tumors and improves survival (p < 0.05). Addition of αPD-1 to SBRT is associated with increases in CD8+ cytotoxic T cells based on flow cytometry (Figure ​(Figure1a)1a) and CD3+ T cells based on six-color immunofluorescence, and this augmentation of immune response is associated with increased ICOS expression on T cells within tumor and spleen. Importantly, the tumors grow out rapidly after αPD-1 is discontinued. The percentage of regulatory T cells (CD4+CD25+FOXP3+) also increases significantly after αPD-1 alone and in combination with SBRT (p < 0.001), suggesting a potential mechanism by which cytotoxic T cells could be rendered anergic. In addition, the combination of αPD-1 plus SBRT induced programmed cell death-1 ligand expression on suppressive macrophages based on flow cytometry (Figure ​(Figure1b)1b) and immunofluorescence, suggesting another potential mechanism of resistance to long term immune control of the tumors. Conclusion: Tumor response to SBRT can be augmented by concurrent administration of αPD-1 antibody. The efficacy of this combination therapy is transient, however. The environment of chronic immune suppression in most patients affected by HCC adds to the rationale of this immune-radiotherapy combination, but the mechanisms of immune resistance will need to be overcome for this combination to generate lasting immunity. Figure 1

Targeting Immune Suppressive Microenvironment By Immune Checkpoint Blockade in Multiple Myeloma

Bidirectional interaction between MM cells and accessory cells regulates tumor development on the one hand, while transforming the BM microenvironment into a tumor promoting and immune suppressive milieu on the other. Recent developments in targeted therapies have indicated that generation of the most effective therapeutic strategies requires not only targeting tumor or stroma, but also methods to overcome blockade of anti-tumor immune responses. Tumor associated immune suppressor cells such as Treg and myeloid derived suppressor cells (MDSC) can effectively block anti-tumor immune responses, thereby representing an important obstacle for immunotherapy. Co-inhibitory molecules programmed cell death-1 (PD-1) and its ligand (PD-L1) play a fundamental role in tumor immune escape by inhibiting immune effector functions. Since PD-1/PD-L1 signaling promotes tumor growth while inhibiting effector cell mediated anti-tumor immune responses, we here assessed the impact of PD-1/PD-L1 blockade, alone or in combination with lenalidomide (Len), on accessory and immune cells function, as well as tumor cell growth of MM in the BM milieu.Methods: PD-L1 gene expression was determined in IFM MM data set (n=170). Cell surface expression of PD-L1 in CD138+ MM cells, stroma, and MDSCs; as well as PD-1 expression on effector cells (CD4T, CD8T, NK, NKT and monocytes/macrophages) were determined in the bone marrow (BM) and peripheral blood (PB) of patients with MGUS (n=3), newly diagnosed (ND-MM, n=5) and relapsed/refractory (RR-MM, n=11) compared to healthy donors (HD, n=10). PD-1/PD-L1 signaling was assessed in the autologous cocultures of patient MM cells or MM cell lines with stroma (BMSC) or MDSCs and effector cells, in the presence or absence of PD-1/PD-L1 blockade and Len. Len effect on PD-1 expression on effector cells and PD-L1 expression on tumor, BMSC and MDSCs was determined by flow cytometry analysis. BMSC and MDSC induced MM growth/viability was measured by MTT, 3H-Thy and CFSE flow cytometry analysis. Effector cell-mediated MM cytotoxicity was measured by CFSE/PI flow cytometry. The effect of PD-1/PD-L1 blockade with or without Len on cytokine pattern was determined by intracellular cytokine flow cytometry analysis.Results: Statistical analysis of IFM MM data demonstrated that the majority of patient MM cells have increased PD-L1 mRNA compared to HD (p=0.0064). Cell surface expression of PD-L1 was also significantly increased in ND-MM cells (median 65%) and even higher in RR-MM cells. Correspondingly, there was a significant increase in PD-1 expression on CD8T and NK cells in ND-MM and RR-MM. Moreover, PD-L1 expression was significantly higher on both mMDSC and nMDSC than APCs in ND-MM and RR-MM. Coculture with BMSC significantly increased expression of PD-L1 on MM cells. PD-1/ PD-L1 blockade overcame BMSC-induced tumor cell growth in both patient MM cells and MM cell lines. Importantly, Len significantly reduced PD-L1 expression on MM cells; and combined blockade of PD-1/PD-L1 with Len further decreased BMSC-induced MM growth. Immunomodulatory effects of PD-1/PD-L1 blockade were also evaluated in autologous cocultures of immune effector cells with MM cells. Even though there was no change in effector cell proliferation, PD-1/PD-L1 blockade significantly induced cytotoxic activity of autologous T cells, NK cells, and macrophages cultured with MM cells; and Len further enhanced effector cell-mediated cytotoxicity. PD-1/PD-L1 blockade induced intracellular expression of cytotoxic cytokines IFNg and Granzyme B (Gzm B) in CD4T cells, CD8T cells, NK cells and Macrophages. Furthermore, MDSC-mediated MM growth was significantly decreased by PD-1/ PD-L1 blockade. Finally, PD-1/PD-L1 blockade induced intracellular expression of IFNg and Gzm B in T cells, NK cells and NKT cells cultured with autologous MDSC; and Len further enhanced this effector cell activation.Conclusion: Our data demonstrated that immune checkpoint signaling plays an important role in providing the tumor promoting, immune suppressive microenvironment in MM. Blockade of PD-1/PD-L1 signaling induces anti-MM immune responses that can be enhanced by Len. Targeting checkpoint signaling using PD-1 and PD-L1 blocking antibodies, particularly in combination with Len, therefore represents a promising novel immune-based therapeutic strategy to both inhibit tumor cell growth and restore host immune function in MM. DisclosuresKikuchi:The ITO Foundation for the Promotion of Medical Science: Research Funding. Hideshima:Acetylon Pharmaceuticals: Consultancy. Raje:novartis, Amgen, Celgene, Millenium, Onyx: Consultancy; Eli Lilly, Acetylon: Research Funding. Anderson:Celgene: Consultancy; Onyx: Consultancy; Gilead Sciences: Consultancy; Sanofi-Aventis US: Consultancy; Acetylon: Scientific Founder Other; Oncoprep: Scientific Founder Other.

Nivolumab in Patients with Relapsed or Refractory Hodgkin Lymphoma - Preliminary Safety, Efficacy and Biomarker Results of a Phase I Study

Introduction: Classical Hodgkin lymphoma (cHL) is characterized by Reed Sternberg (RS) cells surrounded by an extensive but ineffective inflammatory/immune cell infiltrate. Recent studies suggest that Hodgkin RS cells have developed mechanisms that exploit the programmed cell death-1 (PD-1) pathway to evade immune detection. In cHL, chromosome 9p24.1 gain is a frequent structural alteration that increases the gene dosage of the PD-1 ligands, PD-L1 and PD-L2, and their induction via JAK/STAT signaling. Epstein-Barr Virus (EBV) infection also increases the expression of the PD-1 ligands in EBV-positive cHL. Ligand binding to PD-1 receptor-positive activated T cells induces “T cell exhaustion,” a reversible inhibition of T cell activation and proliferation.Nivolumab (BMS-936558, Bristol-Myers Squibb) is a fully human IgG4 monoclonal PD-1 blocking antibody that potentiates anti-tumor T cell activity, and exhibits clinical efficacy in several solid tumors. We hypothesized that nivolumab may augment anti-tumor activity in patients with relapsed or refractory (R/R) cHL, including those who had failed brentuximab vedotin (BV), and evaluated the PD-1 blocking antibody. These are the first analyses being reported with PD-1 antibody for the treatment of cHL.Methods: Patients with R/R cHL were included as an independent cohort in a dose escalation and cohort expansion phase I study of nivolumab in lymphoma and multiple myeloma (MM). Results for patients with non-Hodgkin lymphoma and MM are reported separately. Patients with cHL received nivolumab 3 mg/kg every 2 weeks until confirmed tumor progression or excessive toxicity. Responses were evaluated using standard criteria. The primary endpoint was safety; key secondary endpoints included anti-tumor activity and expression of immunomodulatory proteins in tumor biopsies.Results: Twenty-three patients were enrolled with R/R cHL. Patients were heavily pre-treated, 87% had received ≥ 3 prior treatment regimens, 78% had prior autologous stem cell transplant (ASCT), and 78% had prior BV treatment. Drug-related adverse events (AEs) of any grade occurred in 78% of patients, the most common of which were rash (22%), decreased platelet count (17%), diarrhea, nausea, pruritus, fatigue, and pyrexia (each at 13%). Drug-related grade 3/4 AEs occurred in 22% of patients. Three patients experienced 1 serious AE each (grade 3 myelodysplastic syndrome [MDS], grade 3 pancreatitis, and grade 2 lymph node pain).The objective response rate (ORR) was 87% (20/23), with 4 patients (17%) achieving a complete response (CR) and 16 (70%) obtaining a partial response (PR). The remaining 3 patients (13%) had stable disease (SD). All 23 patients had a reduction in tumor burden at 1 or more efficacy assessments during treatment with nivolumab (Figure). Among the 18 patients who had previously failed BV, the ORR was 89% (16/18), with 6% (1/18) achieving CR and 83% (15/18) PR. Progression-free survival (PFS) rate at 24 weeks was 86% (95% CI, 62-95%). The median overall survival (OS) has not been reached (range, 21+ to 75+ weeks). Six of 23 patients elected to discontinue study treatment to undergo stem cell transplantation, 2 patients discontinued due to toxicities (MDS and thrombocytopenia in 1 patient; pancreatitis in 1 patient), 4 patients discontinued due to disease progression, and 11 patients continue on study as of June 16, 2014.In a subset of study patients (10/23), PD-L1 and PD-L2 copy numbers in RS cells were assessed using fixed tumor biopsy specimens and a 3-probe fluorescence in situ assay (PD-L1,PD-L2, and control centromeric probe). In all tumors, RS cells had copy gains of PD-L1 and PD-L2, as a result of either polysomy or amplification; these tumors also exhibited increased protein expression of PD-L1. RS cells were also largely positive for pSTAT3, indicative of active JAK/STAT signaling.Conclusions In patients with R/R cHL, nivolumab-mediated PD-1 blockade is safe and tolerable with a safety profile similar to that in solid tumors. The frequent and long-lasting responses in heavily pretreated, R/R patients, including those who have failed BV, highlight the importance of the PD-1 pathway in cHL, and the genetically defined sensitivity to PD-1 blockade in this disease. Based on these results, the FDA granted nivolumab breakthrough status in relapsed cHL and a large, multinational, phase II trial of this therapy is underway. [Display omitted] DisclosuresArmand:Merck: Consultancy. Off Label Use: Nivolumab was tested for the treatment of hematologic malignancies.. Ansell:Bristol-Myers Squibb: Research Funding. Lesokhin:Bristol-Myers Squibb: Consultancy, Research Funding. Timmerman:Bristol-Myers Squibb: Research Funding. Borrello:Bristol-Myers Squibb: Research Funding. Rodig:Bristol-Myers Squibb: Research Funding. Zhu:Bristol-Myers Squibb: Employment. Grosso:Bristol-Myers Squibb: Employment, Equity Ownership. Kim:Bristol-Myers Squibb: Employment. Shipp:Merck: Membership on an entity’s Board of Directors or advisory committees; Pharmacyclics: Membership on an entity’s Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Bayer: Membership on an entity’s Board of Directors or advisory committees; Sanofi: Research Funding; Janssen R&D: Membership on an entity’s Board of Directors or advisory committees.

Blockade of PD1 and TIM3 Restores Innate and Adaptive Immunity in Patients With Acute Alcoholic Hepatitis

Susceptibility to bacterial infection is a feature of alcohol-related liver disease. Programmed cell death 1 (PD1), the T-cell immunoglobulin and mucin domain-containing protein 3 (TIM3, also known as hepatitis A virus cellular receptor 2), and their respective ligands-CD274 (also known as PD ligand 1 [PDL1]) and galectin-9-are inhibitory receptors that regulate the balance between protective immunity and host immune-mediated damage. However, their sustained hyperexpression promotes immune exhaustion and paralysis. We investigated the role of these immune inhibitory receptors in driving immune impairments in patients with alcoholic liver disease. In a prospective study, we collected blood samples from 20 patients with acute alcoholic hepatitis (AAH), 16 patients with stable advanced alcohol-related cirrhosis, and 12 healthy individuals (controls). Whole blood or peripheral blood mononuclear cells were assessed for expression of PD1, PDL1, TIM3, galectin-9, and Toll-like receptors on subsets of innate and adaptive immune effector cells. We measured antibacterial immune responses to lipopolysaccharide (endotoxin) using ELISpot assays, and used flow cytometry to quantify cytokine production, phagocytosis, and oxidative burst in the presence or absence of blocking antibodies against PD1 or TIM3. Antibacterial innate and adaptive immune responses were greatly reduced in patients with AAH, compared with controls, and patients with alcohol-related cirrhosis had less severe dysfunctions in innate immune effector cells and preserved functional T-cell responses. Fewer T cells from patients with AAH produced interferon gamma in response to lipopolysaccharide, compared with controls. In addition, patients with AAH had greater numbers of interleukin 10-producing T cells, and reduced levels of neutrophil phagocytosis and oxidative burst in response to Escherichia coli stimulation, compared with controls. T cells from patients with AAH, but not alcohol-related cirrhosis, expressed higher levels of PD1 and PDL1, or TIM3 and galectin-9, than T cells from controls. Antibodies against PD1 and TIM3 restored T-cell production of interferon gamma, reduced the numbers of interleukin 10-producing T cells, and increased neutrophil antimicrobial activities. Circulating levels of endotoxin in plasma from patients with AAH caused over expression of immune inhibitory receptors on T cells via Toll-like receptor 4 binding to CD14(+) monocytes. Antibacterial immune responses are impaired in patients with AAH. Lymphocytes from these patients express high levels of immune inhibitory receptors, produce lower levels of interferon gamma, and have increased IL10 production due to chronic endotoxin exposure. These effects can be reversed by blocking PD1 and TIM3, which increase the antimicrobial activities of T cells and neutrophils.

Harnessing the PD-1 pathway in renal cell carcinoma: current evidence and future directions.

Programmed cell death-1 (PD-1) is a recognized immune checkpoint. It is frequently upregulated on the T cells that infiltrate tumors, providing an inhibitory signal, which may facilitate immune escape. Blocking antibodies have been developed to interrupt the interaction of PD-1 with its ligands PD-L1/PD-L2, with the goal of increasing the host antitumor immune response. Initial results have been encouraging, with durable responses in both treatment-naive and pretreated patients, along with an acceptable toxicity profile. This tolerability makes PD-1 blockade an excellent potential partner for combination strategies with the approved targeted agents, such as tyrosine kinase inhibitors (TKIs) and anti-vascular endothelial growth factor (anti-VEGF) antibodies, as well as other investigational immune checkpoint inhibitors or agonist antibodies that may costimulate an immune response. PD-L1 expression on tumor cells and tumor-infiltrating immune cells is also being evaluated as a predictive biomarker of response to treatment. This review summarizes the biological basis, preclinical studies, ongoing trials, and future challenges associated with targeting the PD-1 pathway in renal cell carcinoma.

Abstract 2810: Dendritic cell-based immunotherapy in combination with programmed cell death 1 blockade reduced established tumors by activating Th-1 type immune responses in a murine hepatocellular carcinoma model

Abstract Progress in treatments for hepatocellular carcinoma (HCC) has improved the prognosis of patients with HCC. However, HCC is usually associated with cirrhosis and often recurs even after complete treatment of the tumors in the remaining part of the cirrhotic liver. Thus, there is a strong need for the development of a new intervention therapy that suppresses the occurrence or recurrence effectively with fewer side effects. Immunotherapy may be such a treatment and several clinical trials have been performed for HCC treatment. Dendritic cells (DCs) are known as professional antigen-presenting cells characterized by their potent ability to elicit immune responses to tumor-specific T cells against tumor-associated antigens. Programmed cell death 1 (PD-1) has been identified as a marker of exhausted T cells in chronic disease states, and blockade of PD-1-PD-ligand interactions has been shown to partially restore T cell function. In this study, we evaluated the efficacy of the combination of DC-based vaccine and PD-1 blockade, and investigated the mechanisms of the antitumor effects of the combined therapy. In protection model, mice were injected with DCs or/and PD-1-antibody before the murine HCC tumor cell (BNL cell) challenge. 40% of mice treated with both DCs and PD-1-antibody rejected tumor challenge, whereas other groups observed a palpable tumor in all mice tested. In therapeutic model, tumor-bearing mice were inoculated with DCs or/and PD-1-antibody. Significant suppression of outgrowth of the established tumors was observed in the DCs and anti-PD-1 combination treatment group (DCs + anti-PD-1, 138.00 ± 56.66 mm2 vs control, 402.33 ± 40.63 mm2 on day 42, P = 0.0073 vs controls). Immunohistochemical analyses of therapeutic model showed marked infiltration of CD4+ cells, CD8+ cells and CD11c+ cells in the established BNL tumors of mice treated with both DCs and PD-1-antibody. To investigate induction of tumor-specific immune responses, we stimulated splenocytes of DCs or/and PD-1-antibody treated mice twice weekly by DCs in vitro. Significant tumor-specific cytolysis was detected when splenocytes of mice treated with both DCs and anti-PD-1 were used as effector cells (30.0% ± 2.8% for BNL and 7.3% ± 0.1% for YAC-1, effector to target ratio; E/T=40). Our findings suggest that blockade of the PD-1 PD-ligand enhanced the Th1-type antitumor immune responses induced by DC vaccine. The combination of DC vaccine and PD-1 blockade may be a possible candidate for a cancer vaccine for clinical trials. Citation Format: Eiichi Hayashi, Junichi Eguchi, Masashi Sakaki, Hiroyoshi Doi, Risa Oomori, Atsushi Kajiwara, Hitoshi Yoshida, Shigeaki Ishii, Kazumasa Hiroishi, Michio Imawari. Dendritic cell-based immunotherapy in combination with programmed cell death 1 blockade reduced established tumors by activating Th-1 type immune responses in a murine hepatocellular carcinoma model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2810. doi:10.1158/1538-7445.AM2014-2810

Abstract 5020: Cooperative effects of programmed cell death-1 blockade and radiation in a model of a poorly immunogenic breast carcinoma

Abstract We have previously shown that radiation therapy (RT) converts poorly immunogenic tumors resistant to antibodies (mAbs) against the checkpoint receptor CTLA-4 into susceptible ones. Importantly, RT induced the upregulation of PDL-1 and PDL-2 on the tumor cells and infiltrating myeloid cells in the TSA mouse breast cancer, suggesting that PD-1 ligands may limit rejection of the irradiated tumor by T cells. PD-1 is a checkpoint receptor upregulated on T cells shortly after activation and expressed at high levels on exhausted T cells. To test the hypothesis PD-1 blockade can promote the ability of RT to induce effective anti-tumor immunity we employed BALB/c mice inoculated s.c. with the syngeneic poorly immunogenic TSA breast cancer cells. When tumors became palpable mice were randomly assigned to one of 4 treatment groups (N=6-7): (1) control, (2) RT, (3) anti-PD-1 mAb and (4) RT+anti-PD-1 mAb. RT was delivered exclusively to the primary tumor in 8 Gy fractions on days 13, 14 and 15 post tumor inoculation. PD-1 blocking mAb RMP1-14 was given on day 15 and every 4 days thereafter, and mice were followed for tumor growth. In a separate experiment, mice were euthanized on day 20 to characterize tumor-infiltrating lymphocytes (TILs) and development of CD8+ T cells specific for the tumor epitope AH1 by pentamer analysis. TSA tumors were resistant to PD-1 blockade. RT significantly delayed tumor growth (p&amp;lt;0.01), but tumor regression was seen only in 1 of 6 mice. In contrast, when RT was combined with anti-PD-1 mAb, all of the mice completely rejected tumors by day 25. AH1-specific CD8+ T-cells were markedly increased (5.5%) in the spleen of mice treated with RT+anti-PD-1 compared to control (1.7%) and anti-PD-1 (1.9%) or RT (3.7%) treated mice (p&amp;lt;0.01). Analysis of TILs showed a significant increase in mice treated with RT compared to control in CD8+ T cells expressing high levels of PD-1 (CD8+PD-1hi) (67% versus 36%, p&amp;lt;0.01), as detected by a mAb non-cross-reactive with RMP1-14. Interestingly, in mice receiving anti-PD-1 mAb there was a significant decrease in CD8+PD-1hi (14% RMP1-14 versus 36% control, p&amp;lt;0.01; and 30% RT+RMP1-14 versus 67% RT, p&amp;lt;0.01). In contrast, CD8+ T cells expressing activation markers CD69 and CD137 and low levels of PD-1 were increased significantly in the tumors of mice treated with RT (64%) compared to control (42%) (p&amp;lt;0.001) irrespective of the treatment with anti-PD-1. Overall, data suggest that the IgG2a anti-PD-1 mAb RMP1-14 selectively depletes CD8+PD-1hi TILs, possibly by ADCC, while enhancing the RT-elicited priming and activation of tumor-specific CD8+ T cells. We propose that this represents a novel mechanism of anti-tumor activity of anti-PD-1 mAb and supports testing its combination with radiotherapy. Supported by the Breast Cancer Alliance (Exceptional Project Award). Citation Format: Karsten Pilones, Joseph Aryankalayil, Silvia Formenti, Sandra Demaria. Cooperative effects of programmed cell death-1 blockade and radiation in a model of a poorly immunogenic breast carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5020. doi:10.1158/1538-7445.AM2014-5020

VISTA Is an Immune Checkpoint Molecule for Human T Cells

V-domain Ig suppressor of T cell activation (VISTA) is a potent negative regulator of T-cell function that is expressed on hematopoietic cells. VISTA levels are heightened within the tumor microenvironment, in which its blockade can enhance antitumor immune responses in mice. In humans, blockade of the related programmed cell death 1 (PD-1) pathway has shown great potential in clinical immunotherapy trials. Here, we report the structure of human VISTA and examine its function in lymphocyte negative regulation in cancer. VISTA is expressed predominantly within the hematopoietic compartment with highest expression within the myeloid lineage. VISTA-Ig suppressed proliferation of T cells but not B cells and blunted the production of T-cell cytokines and activation markers. Our results establish VISTA as a negative checkpoint regulator that suppresses T-cell activation, induces Foxp3 expression, and is highly expressed within the tumor microenvironment. By analogy to PD-1 and PD-L1 blockade, VISTA blockade may offer an immunotherapeutic strategy for human cancer.

Enhancing Virus-Specific Immunity In Vivo by Combining Therapeutic Vaccination and PD-L1 Blockade in Chronic Hepadnaviral Infection

Hepatitis B virus (HBV) persistence is facilitated by exhaustion of CD8 T cells that express the inhibitory receptor programmed cell death-1 (PD-1). Improvement of the HBV-specific T cell function has been obtained in vitro by inhibiting the PD-1/PD-ligand 1 (PD-L1) interaction. In this study, we examined whether in vivo blockade of the PD-1 pathway enhances virus-specific T cell immunity and leads to the resolution of chronic hepadnaviral infection in the woodchuck model. The woodchuck PD-1 was first cloned, characterized, and its expression patterns on T cells from woodchucks with acute or chronic woodchuck hepatitis virus (WHV) infection were investigated. Woodchucks chronically infected with WHV received a combination therapy with nucleoside analogue entecavir (ETV), therapeutic DNA vaccination and woodchuck PD-L1 antibody treatment. The gain of T cell function and the suppression of WHV replication by this therapy were evaluated. We could show that PD-1 expression on CD8 T cells was correlated with WHV viral loads during WHV infection. ETV treatment significantly decreased PD-1 expression on CD8 T cells in chronic carriers. In vivo blockade of PD-1/PD-L1 pathway on CD8 T cells, in combination with ETV treatment and DNA vaccination, potently enhanced the function of virus-specific T cells. Moreover, the combination therapy potently suppressed WHV replication, leading to sustained immunological control of viral infection, anti-WHs antibody development and complete viral clearance in some woodchucks. Our results provide a new approach to improve T cell function in chronic hepatitis B infection, which may be used to design new immunotherapeutic strategies in patients.

Safety and activity of PD1 blockade by pidilizumab in combination with rituximab in patients with relapsed follicular lymphoma: a single group, open-label, phase 2 trial

Endogenous or iatrogenic antitumour immune responses can improve the course of follicular lymphoma, but might be diminished by immune checkpoints in the tumour microenvironment. These checkpoints might include effects of programmed cell death 1 (PD1), a co-inhibitory receptor that impairs T-cell function and is highly expressed on intratumoral T cells. We did this phase 2 trial to investigate the activity of pidilizumab, a humanised anti-PD1 monoclonal antibody, with rituximab in patients with relapsed follicular lymphoma. We did this open-label, non-randomised trial at the University of Texas MD Anderson Cancer Center (Houston, TX, USA). Adult (≥18 years) patients with rituximab-sensitive follicular lymphoma relapsing after one to four previous therapies were eligible. Pidilizumab was administered at 3 mg/kg intravenously every 4 weeks for four infusions, plus eight optional infusions every 4 weeks for patients with stable disease or better. Starting 17 days after the first infusion of pidilizumab, rituximab was given at 375 mg/m(2) intravenously weekly for 4 weeks. The primary endpoint was the proportion of patients who achieved an objective response (complete response plus partial response according to Revised Response Criteria for Malignant Lymphoma). Analysis was by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT00904722. We enrolled 32 patients between Jan 13, 2010, and Jan 20, 2012. Median follow-up was 15.4 months (IQR 10.1-21.0). The combination of pidilizumab and rituximab was well tolerated, with no autoimmune or treatment-related adverse events of grade 3 or 4. The most common adverse events of grade 1 were anaemia (14 patients) and fatigue (13 patients), and the most common adverse event of grade 2 was respiratory infection (five patients). Of the 29 patients evaluable for activity, 19 (66%) achieved an objective response: complete responses were noted in 15 (52%) patients and partial responses in four (14%). The combination of pidilizumab plus rituximab is well tolerated and active in patients with relapsed follicular lymphoma. Our results suggest that immune checkpoint blockade is worthy of further study in follicular lymphoma. National Institutes of Health, Leukemia and Lymphoma Society, Cure Tech, and University of Texas MD Anderson Cancer Center.

Therapeutic PD-1 Pathway Blockade Augments with Other Modalities of Immunotherapy T-Cell Function to Prevent Immune Decline in Ovarian Cancer

The tumor microenvironment mediates induction of the immunosuppressive programmed cell death-1 (PD-1) pathway, and targeted interventions against this pathway can help restore antitumor immunity. To gain insight into these responses, we studied the interaction between PD-1 expressed on T cells and its ligands (PD-1:PD-L1, PD-1:PD-L2, and PD-L1:B7.1), expressed on other cells in the tumor microenvironment, using a syngeneic orthotopic mouse model of epithelial ovarian cancer (ID8). Exhaustion of tumor-infiltrating lymphocytes (TIL) correlated with expression of PD-1 ligands by tumor cells and tumor-derived myeloid cells, including tumor-associated macrophages (TAM), dendritic cells, and myeloid-derived suppressor cells (MDSC). When combined with GVAX or FVAX vaccination (consisting of irradiated ID8 cells expressing granulocyte macrophage colony-stimulating factor or FLT3 ligand) and costimulation by agonistic α-4-1BB or TLR 9 ligand, antibody-mediated blockade of PD-1 or PD-L1 triggered rejection of ID8 tumors in 75% of tumor-bearing mice. This therapeutic effect was associated with increased proliferation and function of tumor antigen-specific effector CD8(+) T cells, inhibition of suppressive regulatory T cells (Treg) and MDSC, upregulation of effector T-cell signaling molecules, and generation of T memory precursor cells. Overall, PD-1/PD-L1 blockade enhanced the amplitude of tumor immunity by reprogramming suppressive and stimulatory signals that yielded more powerful cancer control.

Immunotherapy of chronic hepatitis C virus infection with antibodies against programmed cell death-1 (PD-1)

Hepatitis C virus (HCV) persistence is facilitated by exhaustion of CD8+ T cells that express the inhibitory receptor programmed cell death 1 (PD-1). Blockade of PD-1 signaling improves in vitro proliferation of HCV-specific T lymphocytes, but whether antiviral function can be restored in infected individuals is unknown. To address this question, chimpanzees with persistent HCV infection were treated with anti-PD-1 antibodies. A significant reduction in HCV viremia was observed in one of three treated animals without apparent hepatocellular injury. Viremia rebounded in the responder animal when antibody treatment was discontinued. Control of HCV replication was associated with restoration of intrahepatic CD4+ and CD8+ T-cell immunity against multiple HCV proteins. The responder animal had a history of broader T-cell immunity to multiple HCV proteins than the two chimpanzees that did not respond to PD-1 therapy. The results suggest that successful PD-1 blockade likely requires a critical threshold of preexisting virus-specific T cells in liver and warrants consideration of therapeutic vaccination strategies in combination with PD-1 blockade to broaden narrow responses. Anti-PD-1 immunotherapy may also facilitate control of other persistent viruses, notably the hepatitis B virus where options for long-term control of virus replication are limited.

PD-L1 blockade synergizes with IL-2 therapy in reinvigorating exhausted T cells

The inhibitory receptor programmed cell death 1 (PD-1) plays a major role in functional exhaustion of T cells during chronic infections and cancer, and recent clinical data suggest that blockade of the PD-1 pathway is an effective immunotherapy in treating certain cancers. Thus, it is important to define combinatorial approaches that increase the efficacy of PD-1 blockade. To address this issue, we examined the effect of IL-2 and PD-1 ligand 1 (PD-L1) blockade in the mouse model of chronic lymphocytic choriomeningitis virus (LCMV) infection. We found that low-dose IL-2 administration alone enhanced CD8+ T cell responses in chronically infected mice. IL-2 treatment also decreased inhibitory receptor levels on virus-specific CD8+ T cells and increased expression of CD127 and CD44, resulting in a phenotype resembling that of memory T cells. Surprisingly, IL-2 therapy had only a minimal effect on reducing viral load. However, combining IL-2 treatment with blockade of the PD-1 inhibitory pathway had striking synergistic effects in enhancing virus-specific CD8+ T cell responses and decreasing viral load. Interestingly, this reduction in viral load occurred despite increased numbers of Tregs. These results suggest that combined IL-2 therapy and PD-L1 blockade merits consideration as a regimen for treating human chronic infections and cancer.