Host Antitumor Response Research Articles

Integrin β1 activation induces an anti-melanoma host response.

TGF-β is a cytokine thought to function as a tumor promoter in advanced malignancies. In this setting, TGF-β increases cancer cell proliferation, survival, and migration, and orchestrates complex, pro-tumorigenic changes in the tumor microenvironment. Here, we find that in melanoma, integrin β1-mediated TGF-β activation may also produce tumor suppression via an altered host response. In the A375 human melanoma cell nu/nu xenograft model, we demonstrate that cell surface integrin β1-activation increases TGF-β activity, resulting in stromal activation, neo-angiogenesis and, unexpectedly for this nude mouse model, increase in the number of intra-tumoral CD8+ T lymphocytes within the tumor microenvironment. This is associated with attenuation of tumor growth and long-term survival benefit. Correspondingly, in human melanomas, TGF-β1 correlates with integrin β1/TGF-β1 activation and the expression of markers for vasculature and stromal activation. Surprisingly, this integrin β1/TGF-β1 transcriptional footprint also correlates with the expression of markers for tumor-infiltrating lymphocytes, multiple immune checkpoints and regulatory pathways, and, importantly, better long-term survival of patients. These correlations are unique to melanoma, in that we do not observe similar associations between β1 integrin/TGF-β1 activation and better long-term survival in other human tumor types. These results suggest that activation of TGF-β1 in melanoma may be associated with the generation of an anti-tumor host response that warrants further study.

Myeloid-derived suppressor cells and their role in pancreatic cancer.

Pancreatic cancer is a devastating disease and ranks as the third most common cause of cancer-related death. Like many cancers, there has been increased interest in the role of the immune system in the progression and development of pancreatic cancer. In particular, immunosuppression within the tumor microenvironment (TME) is thought to impair the host's antitumor response. In this article, we review myeloid-derived suppressor cells and their contribution to this immunosuppression within the pancreatic TME.

SWI/SNF Chromatin Remodeling Complex Regulates the Expression of PD-L1 in a Spatio-Temporal Manner in Acute Lymphoblastic Leukemia (ALL) in Vitro

Epigenetic changes and interactions between different cells within the tumor microenvironment are critical for the maintenance and progression of cancer. The primary facet of such interactions is to create an immunosuppressive environment for tumor propagation by subverting the host's antitumor response. In this context, one of the critical elements is the Programmed Death 1 (PD-1/CD279), an immune checkpoint protein, which is responsible for the attenuation of the immune response by blocking T-cell function. The interaction between PD-1 on the tumor infiltrating lymphocytes (TILs) and its ligand, PD-L1, on tumor surface is associated with poor prognosis and aggressiveness in several cancers. Interestingly PD-L1 expression on the tumor surface is restricted to regions infiltrated by TILs indicating a direct regulation of PD-L1 - by TIL-secreted proinflammatory cytokines, such as interferon gamma (IFNg) and interleukin 10 (IL-10). This adaptive nature of PD-L1 expression supports the existing concept that epigenetic regulations may serve as a key factor. Nevertheless, there is very little data concerning the PD-L1 expression and the mechanism of immune escape in acute and chronic leukemia.To understand the mechanism of PD-L1 induction in acute leukemia, we evaluated the baseline expression of PD-L1 in four different ALL cell lines, SD1 (pre B), REH (pre B), CCRM-CEM (T-ALL) and MOLT3 (T-ALL). We found that the baseline levels of both PD-L1 mRNA and protein are very low in these cell lines. However, following the treatment with IFNg, we observed a significant increase in PD-L1, both at the mRNA and protein level. Given the fact that IL-15 is required for lymphocyte growth, and is also implicated as an inducer of PD-L1 in solid tumors, we evaluated the effect of IL-15 on PD-L1. We observed an increase in PD-L1 mRNA and protein levels following IL-15 treatment. To gain further insight if epigenetic regulation is involved in these proinflammatory cytokines- mediated PD-L1 upregulation, we studied the role of SWI/SNF complex in this context. SWI/SNF complex is one of the largest, evolutionary conserved chromatin remodeling complex. It contains two catalytic subunits, BRM and BRG1 that have been demonstrated to be dysregulated in different cancers, including leukemia. We found that BRG1 expression in these ALL cell lines goes up significantly following IL-15 and IFNg treatment. To further ascertain if this upregulation of BRG1 is critical for PD-L1 induction, we did an shRNA mediated knockdown of BRG1. This resulted in no PD-L1 induction upon treatment with either IFNg or IL-15 suggesting BRG1 as a downstream effector. This observation is also recapitulated by overexpressing a dominant negative BRG1 variant. Importantly, we also observed that IFNg or IL-15 mediated upregulation of BRG1 is associated with the inactivation of BRM by acetylation. Pharmacological or genetic reversal of BRM acetylation, could inhibit the BRG1-dependent PD-L1 induction following IL-15 or IFNg treatment.To determine how BRM and BRG1 regulate the expression of PD-L1 in the presence of proinflammatory signals, we conducted Chromatin Immunoprecipitation (ChIP) assays on the PD-L1 promoter in the presence or absence of IFNg or IL-15. We found that in the absence of any activating signals (IFNg or IL-15), BRM was recruited to the PD-L1 promoter, along with native STAT1. In the presence of IFNg or IL-15, BRM is acetylated and dissociates from the promoter, along with the recruitment of BRG1 and phosphorylated STAT3, resulting in an increase in PD-L1 transcription.Current immunotherapy involves targeting either PD-1 or PD-L1 by monoclonal antibodies. Although, such therapy has demonstrated promising preliminary results, only a subset of patients has benefited from such therapy in the long term. Furthermore, patients who initially responded to therapy, later developed resistance following relapse. This clinical data suggests that it is imperative to understand the detailed mechanism of PD-L1 regulation to provide better therapeutic options. Our data bestowed a novel insight into the epigenetic regulation of PD-L1 expression on the tumor cells by SWI/SNF chromatin remodeling complex in response to proinflammatory signals. This study provides us with additional therapeutic opportunities, whereby targeting the BRM/BRG1 axis in conjunction with the existing therapy may provide better outcomes. DisclosuresNo relevant conflicts of interest to declare.

PD-L1 expression on neoplastic or stromal cells is respectively a poor or good prognostic factor for adult T-cell leukemia/lymphoma

AbstractProgrammed cell death ligand 1 (PD-L1) is expressed on both tumor and tumor-infiltrating nonmalignant cells in lymphoid malignancies. The programmed cell death 1 (PD-1)/PD-L1 pathway suppresses host antitumor responses, although little is known about the significance of PD-1/PD-L1 expression in the tumor microenvironment. To investigate the clinicopathological impact of PD-L1 expression in adult T-cell leukemia/lymphoma (ATLL), we performed PD-L1 immunostaining in 135 ATLL biopsy samples. We observed 2 main groups: 1 had clear PD-L1 expression in lymphoma cells (nPD-L1(+), 7.4% of patients), and the other showed minimal expression in lymphoma cells (nPD-L1(−), 92.6%). Within the nPD-L1(−) group, 2 subsets emerged: the first displayed abundant PD-L1 expression in nonmalignant stromal cells of the tumor microenvironment (miPD-L1(+), 58.5%) and the second group did not express PD-L1 in any cell (PD-L1(−), 34.1%). nPD-L1(+) ATLL (median survival time [MST] 7.5 months, 95% CI [0.4-22.3]) had inferior overall survival (OS) compared with nPD-L1(−) ATLL (MST 14.5 months, 95% CI [10.1-20.0]) (P = .0085). Among nPD-L1(−) ATLL, miPD-L1(+) ATLL (MST 18.6 months, 95% CI [11.0-38.5]) showed superior OS compared with PD-L1(−) ATLL (MST 10.2 months, 95% CI [8.0-14.7]) (P = .0029). The expression of nPD-L1 and miPD-L1 maintained prognostic value for OS in multivariate analysis (P = .0322 and P = .0014, respectively). This is the first report describing the clinicopathological features and outcomes of PD-L1 expression in ATLL. More detailed studies will disclose clinical and biological significance of PD-L1 expression in ATLL.

Abstract CT053: Intratumoral coxsackievirus A21 increases immune-cell infiltrates and upregulates immune-checkpoint molecules in the tumor microenvironment of advanced melanoma patients: phase II CALM extension study

Abstract Background: CAVATAK, an oncolytic immunotherapy, is a bio-selected oncolytic strain of Coxsackievirus A21 (CVA21). Following intratumoral (IT) injection, CVA21 preferentially infects ICAM-1 expressing tumor cells, resulting in viral replication, cell lysis, and a systemic anti-tumor immune response. The Phase II CALM study investigated the efficacy and safety of IT CVA21 in pts with advanced melanoma with durable responses being observed in both injected and non-injected melanoma metastases, suggesting the generation of significant host anti-tumor responses. Pre-clinical studies in an immune-competent mouse model of melanoma revealed that combinations of intratumoral CAVATAK and anti-PD-1 or anti-CTLA-4 mAbs mediated significantly greater antitumor activity and compared to use of either agent alone. Here we report on an extension study aimed at understanding the impact of CVA21 on immune cell infiltrates and immune checkpoint molecules within treated lesions of advanced melanoma patients. Methods: In the CALM extension study a cohort of 13 advanced melanoma patients received up to 3 × 108 TCID50 CVA21 IT on study days 1,3,5 and 8 and then every three weeks for a further 6 injections. Sequential tumor biopsies of injected lesions (study days 1 and 8) were monitored for evidence of viral-induced changes immune cell infiltrates (Multi-spectral imaging) and immune checkpoint molecules (NanoString® PanCancer Immune profiling panel). Serial serum samples were monitored for viral loads, anti-CVA21 neutralizing antibody (nAb) and levels of immune-inflammatory cytokines. Results: Intratumoral CVA21 administration in a number of cases induced significant increases in immune cell infiltrates within the tumor microenvironment, in particular CD8+ cells (p = 0.044) and increased expression of PD-L1+ cells (p = 0.041) as assessed by multi-spectral imaging. Analysis of a number of pre- and post-treatment biopsy samples by NanoString® digital RNA counting identified notable increases in the levels of immune checkpoint molecules including PD-1, PD-L1, CTLA-4, TIM-3 and LAG-3 and sizable up-regulation of a number of immune modulation elements, including Th1-gene shifts, with increases in interferon-induced genes. Of particular interest was the reconstitution of immune cell infiltrates in a number of CVA21 treated lesions from patients failing previous immune-checkpoint blockade or other immunotherapies. Conclusions: Intratumoral administration of CVA21 can notably influence the dynamics of the tumor micro-environment as evidenced by increases in both immune cell infiltrates and levels of immune-checkpoint genes. Such CVA21 induced immune-changes appear to be beneficial in combination immunotherapy, with preliminary overall response rates in a Phase 1b study CVA21 and anti-CTLA-4 blockade (ipilimumab) in advanced melanoma patients being higher than published response rates for either agent used alone. The use of CVA21 to reconstitute immune cell infiltrates in lesions resistant to immune-checkpoint blockade prior to re-commencing such treatment is an exciting novel therapeutic option. Citation Format: Robert Andtbacka, Brendan Curti, Sigrun Hallmeyer, Zipei Peng, Christopher Paustian, Carlo Bifulco, Bernard Fox, Mark Grose, Darren R. Shafren. Intratumoral coxsackievirus A21 increases immune-cell infiltrates and upregulates immune-checkpoint molecules in the tumor microenvironment of advanced melanoma patients: phase II CALM extension study. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr CT053.

Application of agents of anti-PD-L1 antibody in advanced tumor therapy

Programmed death-1 (PD-1) and its ligand 1 (PD-L1) play critical roles in the identification and elimination of tumor cells evading the host′s immune system. Tumor model in mice which is given anti-PD-L1 monoclonal antibody shows obviously host anti-tumor response. Currently, immunotherapy drugs of PD-1/PD-L1 signaling pathways receive good effects in a variety of tumors failure of the traditional method, and with less adverse reaction, which provide valuable clinical experiences in advanced tumor immunotherapy. Key words: Neoplasms; Immunotherapy; Programmed death ligand 1; Checkpoint inhibitors

Vaccination with Necroptotic Cancer Cells Induces Efficient Anti-tumor Immunity.

Successful immunogenic apoptosis in experimental cancer therapy depends on the induction of strong host anti-tumor responses. Given that tumors are often resistant to apoptosis, it is important to identify alternative molecular mechanisms that elicit immunogenic cell death. We have developed a genetic model in which direct dimerization of FADD combined with inducible expression of RIPK3 promotes necroptosis. We report that necroptotic cancer cells release damage-associated molecular patterns and promote maturation of dendritic cells, the cross-priming of cytotoxic Tcells, and the production of IFN-γ in response to tumor antigen stimulation. Using both FADD-dependent and FADD-independent RIPK3 induction systems, we demonstrate the efficient vaccination potential of immunogenic necroptotic cells. Our study broadens the current concept of immunogenic cell death and opens doors for the development of new strategies in cancer therapy.

Immunobiology of Tumors and Advances in Immunotherapy

Recent studies indicate that functional T-lymphocyte defects compromise anti-tumor responses in various cancers. It appears that tumors not only effectively bypassed the host immune system, but have also actively corrupted the host anti-tumor response via several distinct mechanisms. Cancer Immunotherapy has made significant strides in recent years, due to improved understanding of the underlying principles of tumor biology and immunobiology. This review attempts to identify strategic factors involved in anti-cancer immune impairment, understand the mechanisms underlying reported immunosuppression and highlight some of the most promising present day immunotherapy approaches progressing successfully in clinics.

Tumor-Derived Exosomes and Their Role in Cancer Progression.

Tumor cells actively produce, release, and utilize exosomes to promote tumor growth. Mechanisms through which tumor-derived exosomes subserve the tumor are under intense investigation. These exosomes are information carriers, conveying molecular and genetic messages from tumor cells to normal or other abnormal cells residing at close or distant sites. Tumor-derived exosomes are found in all body fluids. Upon contact with target cells, they alter phenotypic and functional attributes of recipients, reprogramming them into active contributors to angiogenesis, thrombosis, metastasis, and immunosuppression. Exosomes produced by tumors carry cargos that in part mimic contents of parent cells and are of potential interest as noninvasive biomarkers of cancer. Their role in inhibiting the host antitumor responses and in mediating drug resistance is important for cancer therapy. Tumor-derived exosomes may interfere with cancer immunotherapy, but they also could serve as adjuvants and antigenic components of antitumor vaccines. Their biological roles in cancer development or progression as well as cancer therapy suggest that tumor-derived exosomes are critical components of oncogenic transformation.

Expression of Programmed Cell Death 1 and Programmed Cell Death Ligand 1 in Extranodal NK/T-Cell Lymphoma, Nasal Type

BackgroundProgrammed cell death ligand 1 (PD-L1) is expressed on extranodal NK/T-cell lymphoma, nasal type (ENKL) tumor cells. The programmed cell death 1 (PD-1) and PD-L1 pathway inhibits host antitumor responses; however, little is known about how this pathway functions in ENKL. The aim of this study was to investigate the expression of PD-1 and PD-L1 and to determine the clinicopathological impact of PD-1 and PD-L1 positivity in ENKL.MethodsWe performed PD-1 and PD-L1 immunostaining in 79 ENKL biopsy samples and retrospectively analyzed medical records of all 79 patients from 4 tertiary referral hospitals. The demographic features, performance status, stage, LDH, primary sites, nodal sites, hemoglobin, white blood cell, platelet, creatinine, international prognostic index (IPI), and prognostic index for T-cell lymphoma (PIT) were recorded.ResultsThe expression rates of PD-1-positive and PD-L1-positive ENKL were 7.6% and 88.6%, respectively (Figure 1A & 1B). PD-L1-negative ENKL (n=9) was significantly associated with high intermediate or high risk IPI (n=7, P=0.002) and group 3 or 4 PIT (n=6, P=0.043). Patients with PD-1-positive ENKL (n=6) had a trend toward better overall survival (OS) compared with that in patients with PD-1-negative ENKL (P = 0.090, Figure 2A). In contrast, there was no significant difference in OS between PD-L1-positive and -negative ENLK (P = 0.428, Figure 2B).ConclusionsThis is the first report describing the clinicopathological features and survival outcome according to expression of PD-1 and PD-L1 in ENKL. PD-1 expression rate is very low, and PD-L1 negativity is associated with poor risk groups of IPI and PIT in ENKL [Display omitted] [Display omitted] DisclosuresKim:Alexion Pharmaceuticals: Research Funding; Il-Yang: Research Funding; Celgene: Research Funding; Novartis: Research Funding.

Expression of programmed cell death ligand 1 is associated with poor overall survival in patients with diffuse large B-cell lymphoma

AbstractProgrammed cell death ligand 1 (PD-L1) is expressed on both select diffuse large B-cell lymphoma (DLBCL) tumor cells and on tumor-infiltrating nonmalignant cells. The programmed cell death 1 (PD-1)/PD-L1 pathway inhibits host antitumor responses; however, little is known about how this pathway functions in the tumor microenvironment. The aim of this study was to determine the clinicopathological impact of PD-L1+ DLBCL. We performed PD-L1/PAX5 double immunostaining in 1253 DLBCL biopsy samples and established a new definition of PD-L1+ DLBCL. We also defined the criteria for microenvironmental PD-L1+ (mPD-L1+) DLBCL (ie, PD-L1– DLBCL in which PD-L1+ nonmalignant cells are abundant in the tumor microenvironment). Of the 273 patients whose clinical information was available, quantitative analysis of PD-1+ tumor-infiltrating lymphocytes (TILs) was performed. The prevalence rates of PD-L1+ and mPD-L1+ DLBCL were 11% and 15.3%, respectively. Both PD-L1+ and mPD-L1+ DLBCL were significantly associated with non–germinal center B-cell (GCB) type and Epstein-Barr virus positivity. The number of PD-1+ TILs was significantly higher in GCB-type tumors and lower in mPD-L1– and PD-L1+ DLBCL. Patients with PD-L1+ DLBCL had inferior overall survival (OS) compared with that in patients with PD-L1– DLBCL (P = .0009). In contrast, there was no significant difference in OS between mPD-L1+ and mPD-L1– DLBCL (P = .31). The expression of PD-L1 maintained prognostic value for OS in multivariate analysis (P = .0323). This is the first report describing the clinicopathological features and outcomes of PD-L1+ DLBCL. Immunotherapy targeting the PD-1/PD-L1 pathway should be considered in this distinct DLBCL subgroup.

Pseudoprogression and Immune-Related Response in Solid Tumors

Emerging cancer therapeutics target the immune system, stimulating host antitumor response. Tumor cells generate an immunosuppressive milieu with multiple mechanisms to evade immune destruction, including disruption of effective antigen presentation, reduction of effector T-cell function, and upregulation of pathways that promote tolerance and T-cell anergy.1 The programmed death (PD) -1/PD ligand-1 (PD-L1) pathway is a critical component of tumor-mediated immunosuppression. Antibodies to PD-1 and PD-L1 have shown potential clinical benefit in advanced solid tumors.2 The US Food and Drug Administration approved the PD-1 inhibitors pembrolizumab and nivolumab for metastatic melanoma and also recently approved nivolumab for the treatment of metastatic squamous non–small-cell lung cancer. The US Food and Drug Administration has also designated the PD-L1 inhibitor MPDL3280A as a breakthrough therapy for bladder cancer and non–small-cell lung cancer. These drugs and additional immune checkpoint inhibitors are currently under investigation in multiple clinical trials as single-agent therapy and also in combination with other agents.

Toll-like receptor 4 mediates the antitumor host response induced by Ganoderma atrum polysaccharide.

The aim of this study is to investigate the role of Toll-like receptor (TLR) 4 in Ganoderma atrum polysaccharide (PSG-1)-induced antitumor activity. In vitro, the apoptosis rate of S-180 cells was increased in PSG-1-induced peritoneal macrophage derived from C3H/HeN (wild-type) mice, but not from C3H/HeJ (TLR4-deficient) mice. In the S-180 tumor model, phagocytosis, NO and ROS release, phosphorylation of MAPKs and Akt, and expression of NF-κB were increased by PSG-1 in peritoneal macrophage derived from C3H/HeN mice. Furthermore, PSG-1 elevated Th1 cytokine production and enhanced the cytotoxic activity of CTL and NK cells in C3H/HeN mice. In addition, PSG-1 decreased the tumor weight and increased the apoptosis rate and caspase-3 and caspase-9 activities of tumor derived from the C3H/HeN mice. However, none of these activities were observed in C3H/HeJ mice. In summary, these findings demonstrated that the antitumor activity of PSG-1 is mediated by TLR4.

5-Aminolevulinic acid strongly enhances delayed intracellular production of reactive oxygen species (ROS) generated by ionizing irradiation: quantitative analyses and visualization of intracellular ROS production in glioma cells in vitro.

Postoperative adjuvant radiotherapy has important roles in multimodal treatment for highly aggressive malignant gliomas. Previously, we demonstrated that multi-dose ionizing irradiation with repetitive administration of 5-aminolevulinic acid (5-ALA) enhanced the host antitumor response and strongly inhibited tumor growth in experimental glioma. However, the mechanism of the radiosensitizing effect of 5-ALA is not known. Ionizing irradiation not only causes reactive oxygen species (ROS) formation initially by water radiolysis but also induces delayed production of mitochondrial ROS for mediating the long-lasting effects of ionizing irradiation on tumor cells. 5-ALA leads to high accumulation of protoporphyrin IX (PpIX) in the mitochondria of tumor cells, yet can also improve dysfunction of the mitochondrial respiratory chain in tumor cells. Here, we assessed the effect of 5-ALA-induced PpIX synthesis and delayed production of intracellular ROS after ionizing irradiation with 5-ALA in glioma cells in vitro. Temporal changes in intracellular 5-ALA-induced PpIX synthesis after ionizing irradiation in glioma cell lines were evaluated using flow cytometry (FCM). Then, the effect of 5-ALA on delayed production of intracellular ROS 12 h after ionizing irradiation in glioma cells was evaluated by FCM and confocal laser scanning microscopy. Ionizing irradiation had no effect on 5-ALA-induced PpIX synthesis in glioma cells. Delayed intracellular production of ROS was significantly higher than that just after ionizing irradiation, but 5-ALA pretreatment strongly enhanced the delayed intracellular production of ROS, mainly in the cytoplasm of glioma cells. This 5-ALA-induced increase in the delayed production of ROS tended to be higher in the case of 5-ALA treatment before rather than after ionizing irradiation. These results suggest that 5-ALA can affect tumor cells under ionizing irradiation, and greatly increase secondary intracellular production of ROS long after ionizing irradiation, thereby causing a radiosensitizing effect in glioma cells.

5-aminolevulinic acid-induced protoporphyrin IX with multi-dose ionizing irradiation enhances host antitumor response and strongly inhibits tumor growth in experimental glioma in vivo.

Ionizing irradiation is a well‑established therapeutic modality for malignant gliomas. Due to its high cellular uptake, 5‑aminolevulinic acid (ALA) is used for fluorescence‑guided resection of malignant gliomas. We have previously shown that 5‑ALA sensitizes glioma cells to irradiation in vitro. The aim of the present study was to assess whether 5‑ALA acts as a radiosensitizer in experimental glioma in vivo. Rats were subcutaneously injected with 9L gliosarcoma cells and administered 5‑ALA. The accumulation of 5‑ALA‑induced protoporphyrin IX was confirmed by high‑performance liquid chromatography (HPLC) analysis. Subcutaneous (s.c.) tumors were subsequently irradiated with 2 Gy/day for five consecutive days. In the experimental glioma model, high‑performance liquid chromatography analysis revealed a high level of accumulation of 5‑ALA‑induced protoporphyrin IX in s.c. tumors 3 h after 5‑ALA administration. Multi‑dose ionizing irradiation induced greater inhibition of tumor growth in rats that were administered 5‑ALA than in the non‑5‑ALA‑treated animals. Immunohistochemical analysis of the s.c. tumors revealed that numerous ionized calcium‑binding adapter molecule 1 (Iba1)‑positive macrophages gathered at the surface of and within the s.c. tumors following multi‑dose ionizing irradiation in combination with 5‑ALA administration. By contrast, the s.c. tumors in the control group scarcely showed aggregation of Iba1‑positive macrophages. These results suggested that multi‑dose ionizing irradiation with 5‑ALA induced not only a direct cytotoxic effect but also enhanced the host antitumor immune response and thus caused high inhibition of tumor growth in experimental glioma.

Unleashing the immune system: PD-1 and PD-Ls in the pre-treatment tumor microenvironment and correlation with response to PD-1/PD-L1 blockade

Focal tumor cell PD-L1 expression adjacent to TIL can be used as a surrogate marker of an ongoing antitumor host response, which may be unleashed by PD-1 blockade. Tumor cell PD-L1 expression is superior to TIL PD-1 expression and the presence of TIL alone, when predicting response to anti-PD-1 therapy.

Abstract 2939: CAVATAK-mediated oncolytic immunotherapy in advanced melanoma patients

Abstract Background: Coxsackievirus A21 (CVA21) is a naturally occurring “common cold” intercellular adhesion molecule-1 (ICAM-1) targeted RNA virus. Surface ICAM-1 is up-regulated on a number of cancers including melanoma, non-small cell lung, bladder, breast and prostate cancers. CAVATAK is a novel bio-selected formulation of CVA21, which displays potent oncolytic activity against in vitro cultures of cancer cells and in vivo xenografts of a number of cancers. In addition, in vivo tumor challenge studies in immune-competent mouse models have shown that CVA21 lysed tumor cells can induce a secondary systemic host-generated anti-tumor immune response. In this report, advanced melanoma patients were administered multiple intralesional injections of CAVATAK and monitored for markers of host anti-tumor activity on metastatic non-injected lesions. Methods: The Phase II CALM (CAVATAK in Late stage Melanoma) study is investigating the efficacy and safety of intratumoral CAVATAK in 54 advanced melanoma patients. Patient tumors are injected with up to 3 x 10e8 TCID50 CAVATAK on study days 1, 3, 5, 8 and 22 then every 3 weeks for a further 5 injections. CAVATAK-mediated immune anti-tumor activity was assessed by monitoring reductions in non-injected lesions (CT-scan and callipers) and assessing the relative serum levels of a panel of immune inflammatory cytokines. In addition, levels of serum CAVATAK and anti-CAVATAK neutralizing antibodies were assessed on multiple occasions prior to intralesional injections. Results: Intralesional CAVATAK injections resulted in significant responses in a number of melanoma lesions. However, in addition to melanoma tumor responses directly from CAVATAK-mediated oncolysis, a number of non-injected metatastic lesions displayed significant reductions or even complete destruction. Such anti-tumor activity in non-injected lesions is highly suggestive of being mediated via a host generated anti-tumor immune response. Such responses have occurred at times when no circulating infectious CAVATAK was detected in patient serum and in an environment of high levels of anti-CAVATAK neutralizing antibody. In further support of the generation of CAVATAK-mediated immune anti-tumor activity is the identification a possible novel serum cytokine signature of elevated levels of IL-8 and γ-IFN in treated patients linked to systemic tumor response. IL-8 is known to be secreted by activated macrophages, while γ-IFN is produced by stimulated cytotoxic T-cells / NK cells. Conclusion: With a number of patients in the Phase II CALM study displaying reductions in non-injected lesions, together with elevated serum levels of the immune inflammatory cytokines IL-8 and γ-IFN, at times with no detectable levels of circulating infectious CAVATAK, we conclude the generation of a targeted CAVATAK-mediated host anti-tumor response acting on metastatic melanoma lesions is highly likely. Clinical trial information: NCT01227551. Citation Format: Robert H. Andtbacka, Darren R. Shafren, Mark Grose, Len Post, Jeffrey Weisberg. CAVATAK-mediated oncolytic immunotherapy in advanced melanoma patients. [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 2939. doi:10.1158/1538-7445.AM2014-2939

Abstract 2555: A cell based bioluminescent reporter assay for rapid measuring function of PD-1 or PD-L1 therapeutic antibodies

Abstract Programmed death-1(PD-1) and its ligand (PD-L1) are important immunotherapy targets for cancer. PD-1 serves as a negative costimulatory receptor on various cell types, including T and B cells as well as myeloid-derived cells. Its ligand PD-L1 (B7-H1) is not expressed by normal epithelial tissues, but it is aberrantly expressed on a wide array of human cancers including melanoma, non-small cell lung carcinoma, breast cancer, pancreatic cancer, etc. Engagement of PD-1 by its ligands PD-L1 expressed on tumor cells associated with poorer prognosis by disabling the host antitumor response. The most recent investigational anti-PD-1 immunotherapy drugs such as nivolumab from BMS and lambrolizumab (MK-3475) from Merck have all demonstrated significant overall survival rate in patients with advanced melanoma. Here we developed a cell based bioluminescent reporter assay that can be used for rapid quantifying function of PD-1 or PD-L1 therapeutic antibodies as measured by activation of NFAT signaling pathway. For this, Jurkat T-cell line stably expressing NFAT-luciferase reporter and human PD-1 was generated. Raji stably expressing human PD-L1 was generated as antigen presenting cells (APC). by co-cultivating the two cell lines in the present of CD3 and IgG, Raji cells fully activate Jurkat NFAT pathway via CD28 binding to CD80 (B7-1) and CD86 (B7-2) endogenously expressed on Raji cells. PD-1 signaling in Jurkat cells following engagement of PD-L1 ectopically expressed on Raji cells inhibits T cell function, and results in NFAT pathway inhibition. Blockade of PD-L1 using anti-PD-L1 mAb reversed NFAT pathway activation. Citation Format: Mei Cong, Natasha Karassina, Jey Cheng, Frank Fan. A cell based bioluminescent reporter assay for rapid measuring function of PD-1 or PD-L1 therapeutic antibodies. [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 2555. doi:10.1158/1538-7445.AM2014-2555

Impact of non-oncogenic acute viral infection on anti-tumor CD8+ T cell responses (TUM5P.938)

Abstract Background: Factors dictating clinical emergence of cancer are not fully understood. Specifically, how non-oncogenic acute viral infections affect unrelated anti-tumor CD8+ T cell responses (those in uninfected tissues) is unknown. Methods: Therefore, we infected B6 mice with non-lethal Influenza (A/PR/8-OVA, 80,000 EID50, intranasally) 3 days prior to challenge with B16-F10 melanoma (120,000 cells, subcutaneously, right flank). To track self/tumor antigen, virus-expressing antigen, and unrelated foreign antigen CD8+ T cells, we transferred pmel (gp100-specific, Thy1.1+Ly5.1-), OT-I (OVA-specific, Thy1.1+Ly5.1+), and P14 (LCMV gp33, Thy1.1-Ly5.1-) CD8+ T cells, respectively, into Thy1.1-Ly5.1+ B6 mice. Results: Influenza infection significantly worsened normal host anti-tumor responses and decreased host survival with cancer from ~32 to ~15 days (P<0.01). Interestingly, pmel (but not OT-I and P14) CD8+ T cells circulating to the infected lung underwent cell death resulting in their eventual loss from the tumor microenvironment (up to 90%; P<0.001). Similar findings were observed with infections 15 or 30 days before or 7 days after B16 challenge and when Balb/c or humanized NSG mice were challenged with breast cancer or melanoma in the context of LCMV or HIV. Conclusions: Our findings may explain the clinical phenomenon emerging from epidemiologic studies reporting increased unrelated cancer in patients with non-oncogenic viral infections.

HLA-G expression on blasts and tolerogenic cells in patients affected by acute myeloid leukemia.

Human Leukocyte Antigen-G (HLA-G) contributes to cancer cell immune escape from host antitumor responses. The clinical relevance of HLA-G in several malignancies has been reported. However, the role of HLA-G expression and functions in Acute Myeloid Leukemia (AML) is still controversial. Our group identified a subset of tolerogenic dendritic cells, DC-10 that express HLA-G and secrete IL-10. DC-10 are present in the peripheral blood and are essential in promoting and maintaining tolerance via the induction of adaptive T regulatory (Treg) cells. We investigated HLA-G expression on blasts and the presence of HLA-G-expressing DC-10 and CD4+ T cells in the peripheral blood of AML patients at diagnosis. Moreover, we explored the possible influence of the 3′ untranslated region (3′UTR) of HLA-G, which has been associated with HLA-G expression, on AML susceptibility. Results showed that HLA-G-expressing DC-10 and CD4+ T cells are highly represented in AML patients with HLA-G positive blasts. None of the HLA-G variation sites evaluated was associated with AML susceptibility. This is the first report describing HLA-G-expressing DC-10 and CD4+ T cells in AML patients, suggesting that they may represent a strategy by which leukemic cells escape the host's immune system. Further studies on larger populations are required to verify our findings.