Search

Purpose Synovial sarcoma (SySa) is a rare soft tissue tumor characterized by a reciprocal t(X;18) translocation. The chimeric SS18-SSX fusion protein represents the major driver of the disease, acting as aberrant transcriptional dysregulator. Oncogenic mechanisms whereby SS18-SSX mediates sarcomagenesis are incompletely understood, and strategies to selectively target SySa cells remain elusive. Based on results of Phospho-Kinase screening arrays, we here investigate the functional and therapeutic relevance of the transcription factor CREB in SySa tumorigenesis. Methods Immunohistochemistry of phosphorylated CREB and its downstream targets (Rb, Cyclin D1, PCNA, Bcl-xL and Bcl-2) was performed in a large cohort of SySa. Functional aspects of CREB activity, including SS18-SSX driven circuits involved in CREB activation, were analyzed in vitro employing five SySa cell lines and a mesenchymal stem cell model. CREB mediated transcriptional activity was modulated by RNAi-mediated knockdown and small molecule inhibitors (666-15, KG-501, NASTRp and Ro 31-8220). Anti-proliferative effects of the CREB inhibitor 666-15 were tested in SySa avian chorioallantoic membrane and murine xenograft models in vivo. Results We show that CREB is phosphorylated and activated in SySa, accompanied by downstream target expression. Human mesenchymal stem cells engineered to express SS18-SSX promote CREB expression and phosphorylation. Conversely, RNAi-mediated knockdown of SS18-SSX impairs CREB phosphorylation in SySa cells. Inhibition of CREB activity reduces downstream target expression, accompanied by suppression of SySa cell proliferation and induction of apoptosis invitro and in vivo. Conclusion In conclusion, our data underline an essential role of CREB in SySa tumorigenesis and provides evidence for molecular targeted therapies.

CCR Translation for the Article from 2B4 (CD244) Signaling by Recombinant Antigen-specific Chimeric Receptors Costimulates Natural Killer Cell Activation to Leukemia and Neuroblastoma Cells

Purpose: Novel natural killer (NK) cell–directed strategies in cancer immunotherapy aim at specifically modulating the balance between NK cell receptor signals toward tumor-specific activation. The signaling lymphocyte activation molecule–related receptor 2B4 (CD244) is an important regulator of NK cell activation. We investigated whether 2B4-enhanced activation signals can redirect the cytolytic function of human NK cells to NK cell–resistant and autologous leukemia and tumor targets.Experimental Design: In vitro–stimulated NK cells from healthy donors and pediatric leukemia patients were gene modified with CD19 or GD2-specific chimeric receptors containing either the T-cell receptor ζ or 2B4 endodomain alone or combined.Results: Chimeric 2B4 signaling alone failed to induce interleukin-2 receptor up-regulation and cytokine secretion but triggered a specific degranulation response. Integration of the 2B4 endodomain into T-cell receptor ζ chimeric receptors significantly enhanced all aspects of the NK cell activation response to antigen-expressing leukemia or neuroblastoma cells, including CD25 up-regulation, secretion of IFN-γ and tumor necrosis factor-α, release of cytolytic granules, and growth inhibition, and overcame NK cell resistance of autologous leukemia cells while maintaining antigen specificity.Conclusion: These data indicate that the 2B4 receptor has a potent costimulatory effect in NK cells. Antigen-specific 2B4ζ-expressing NK cells may be a powerful new tool for adoptive immunotherapy of leukemia and other malignancies.

Treatments with mono verteporfin and the combination of verteporfin and doxorubicin resulted in a reduction of YAP, TAZ, FOXM1, CTGF and PLK1 protein levels compared to the vehicle control and mono doxorubicin treatment in total protein extracts of a representative set of SySa PDX mice tumors (GAPDH used as loading reference).

Purpose:Synovial sarcoma is a soft tissue malignancy characterized by a reciprocal t(X;18) translocation. The chimeric SS18-SSX fusion protein acts as a transcriptional dysregulator representing the major driver of the disease; however, the signaling pathways activated by SS18-SSX remain to be elucidated to define innovative therapeutic strategies.Experimental Design:Immunohistochemical evaluation of the Hippo signaling pathway effectors YAP/TAZ was performed in a large cohort of synovial sarcoma tissue specimens. SS18-SSX dependency and biological function of the YAP/TAZ Hippo signaling cascade were analyzed in five synovial sarcoma cell lines and a mesenchymal stem cell model in vitro. YAP/TAZ-TEAD–mediated transcriptional activity was modulated by RNAi-mediated knockdown and the small-molecule inhibitor verteporfin. The effects of verteporfin were finally tested in vivo in synovial sarcoma cell line-based avian chorioallantoic membrane and murine xenograft models as well as a patient-derived xenograft.Results:A significant subset of synovial sarcoma showed nuclear positivity for YAP/TAZ and their transcriptional targets FOXM1 and PLK1. In synovial sarcoma cells, RNAi-mediated knockdown of SS18-SSX led to significant reduction of YAP/TAZ-TEAD transcriptional activity. Conversely, SS18-SSX overexpression in SCP-1 cells induced aberrant YAP/TAZ-dependent signals, mechanistically mediated by an IGF-II/IGF-IR signaling loop leading to dysregulation of the Hippo effectors LATS1 and MOB1. Modulation of YAP/TAZ-TEAD–mediated transcriptional activity by RNAi or verteporfin treatment resulted in significant growth inhibitory effects in vitro and in vivo.Conclusions:Our preclinical study identifies an elementary role of SS18-SSX–driven YAP/TAZ signals, highlights the complex network of oncogenic signaling pathways in synovial sarcoma pathogenesis, and provides evidence for innovative therapeutic approaches.

Supplementary Table S1: Clinicopathological characteristics of synovial sarcoma tissue specimens (n=65) and semi-quantitative immunohistochemistry results Supplementary Table S2: Primer sequences

Ewing sarcoma (EwS) is a rare and highly malignant bone tumor occurring mainly in childhood and adolescence. Physiologically, the bone is a central hub for Ca2+ homeostasis, which is severely disturbed by osteolytic processes in EwS. Therefore, we aimed to investigate how ion transport proteins involved in Ca2+ homeostasis affect EwS pathophysiology. We characterized the expression of 22 candidate genes of Ca2+-permeable or Ca2+-regulated ion channels in three EwS cell lines and found the Ca2+-activated K+ channel KCa2.1 (KCNN1) to be exceptionally highly expressed. We revealed that KCNN1 expression is directly regulated by the disease-driving oncoprotein EWSR1-FL1. Due to its consistent overexpression in EwS, KCNN1 mRNA could be a prognostic marker in EwS. In a large cohort of EwS patients, however, KCNN1 mRNA quantity does not correlate with clinical parameters. Several functional studies including patch clamp electrophysiology revealed no evidence for KCa2.1 function in EwS cells. Thus, elevated KCNN1 expression is not translated to KCa2.1 channel activity in EwS cells. However, we found that the low K+ conductance of EwS cells renders them susceptible to hypoosmotic solutions. The absence of a relevant K+ conductance in EwS thereby provides an opportunity for hypoosmotic therapy that can be exploited during tumor surgery.

Ewing sarcoma (EwS) is a rare and highly malignant bone tumor occurring mainly in childhood and adolescence. Physiologically, the bone is a central hub for Ca

Purpose: Synovial sarcoma is a soft tissue malignancy characterized by a reciprocal t(X;18) translocation. The chimeric SS18-SSX fusion protein acts as a transcriptional dysregulator representing the major driver of the disease; however, the signaling pathways activated by SS18-SSX remain to be elucidated to define innovative therapeutic strategies. Experimental Design: Immunohistochemical evaluation of the Hippo signaling pathway effectors YAP/TAZ was performed in a large cohort of synovial sarcoma tissue specimens. SS18-SSX dependency and biological function of the YAP/TAZ Hippo signaling cascade were analyzed in five synovial sarcoma cell lines and a mesenchymal stem cell model in vitro. YAP/TAZ-TEAD–mediated transcriptional activity was modulated by RNAi-mediated knockdown and the small-molecule inhibitor verteporfin. The effects of verteporfin were finally tested in vivo in synovial sarcoma cell line-based avian chorioallantoic membrane and murine xenograft models as well as a patient-derived xenograft. Results: A significant subset of synovial sarcoma showed nuclear positivity for YAP/TAZ and their transcriptional targets FOXM1 and PLK1. In synovial sarcoma cells, RNAi-mediated knockdown of SS18-SSX led to significant reduction of YAP/TAZ-TEAD transcriptional activity. Conversely, SS18-SSX overexpression in SCP-1 cells induced aberrant YAP/TAZ-dependent signals, mechanistically mediated by an IGF-II/IGF-IR signaling loop leading to dysregulation of the Hippo effectors LATS1 and MOB1. Modulation of YAP/TAZ-TEAD–mediated transcriptional activity by RNAi or verteporfin treatment resulted in significant growth inhibitory effects in vitro and in vivo. Conclusions: Our preclinical study identifies an elementary role of SS18-SSX–driven YAP/TAZ signals, highlights the complex network of oncogenic signaling pathways in synovial sarcoma pathogenesis, and provides evidence for innovative therapeutic approaches.

Simple Summary Solid cancers can effectively counteract immune attack by inhibitory checkpoints in the tumor microenvironment. Blockade of relevant immune checkpoints could be a useful tool for enhancing the efficacy of antitumor T cell therapies. Here, we studied the capacity of two nonclassical HLA molecules with known immunosuppressive function, HLA-G and HLA-E, to prevent antigen-specific immune effector functions of gene-engineered T cells against Ewing sarcoma. Inflammatory conditions and interactions of Ewing sarcoma cells with antitumor T cells reliably induced upregulation of the two molecules on the tumor cells. Moreover, as previously shown for HLA-G, HLA-E was detected in a high proportion of human Ewing sarcoma biopsies. However, artificial expression of either of the two molecules on Ewing sarcoma cells failed to reduce cytolytic and activation responses of antigen-specific T cells. We conclude that blockade of HLA-G and HLA-E immune checkpoints is not a promising strategy for enhancing T cell therapies in Ewing sarcoma. Abstract Immune-inhibitory barriers in the tumor microenvironment of solid cancers counteract effective T cell therapies. Based on our finding that Ewing sarcomas (EwS) respond to chimeric antigen receptor (CAR) gene-modified effector cells through upregulation of human leukocyte antigen G (HLA-G), we hypothesized that nonclassical HLA molecules, HLA-G and HLA-E, contribute to immune escape of EwS. Here, we demonstrate that HLA-G isotype G1 expression on EwS cells does not directly impair cytolysis by GD2-specific CAR T cells (CART), whereas HLA-G1 on myeloid bystander cells reduces CART degranulation responses against EwS cells. HLA-E was induced in EwS cells by IFN-γ stimulation in vitro and by GD2-specific CART treatment in vivo and was detected on tumor cells or infiltrating myeloid cells in a majority of human EwS biopsies. Interaction of HLA-E-positive EwS cells with GD2-specific CART induced upregulation of HLA-E receptor NKG2A. However, HLA-E expressed by EwS tumor cells or by myeloid bystander cells both failed to reduce antitumor effector functions of CART. We conclude that non-classical HLA molecules are expressed in EwS under inflammatory conditions, but have limited functional impact on antigen-specific T cells, arguing against a relevant therapeutic benefit from combining CART therapy with HLA-G or HLA-E checkpoint blockade in this cancer.

International audience; Immunotherapy with anti-GD2 monoclonal antibodies (mAbs) provides some benefits for patients with neuroblastoma (NB). However, the therapeutic efficacy remains limited, and treatment is associated with significant neuropathic pain. Targeting O-acetylated GD2 (OAcGD2) by 8B6 mAb has been proposed to avoid pain by more selective tumor cell targeting. Thorough understanding of its mode of action is necessary to optimize this treatment strategy. Here, we found that 8B6-mediated antibody-dependent cellular phagocytosis (ADCP) performed by macrophages is a key effector mechanism. But efficacy is limited by upregulation of CD47 expression on neuroblastoma cells in response to OAcGD2 mAb targeting, inhibiting 8B6-mediated ADCP. Antibody specific for the CD47 receptor SIRPα on macrophages restored 8B6-induced ADCP of CD47-expressing NB cells and improved the antitumor activity of 8B6 mAb therapy. These results identify ADCP as a critical mechanism for tumor cytolysis by anti-disialoganglioside mAb and support a combination with SIRPα blocking agents for effective neuroblastoma therapy.

Common pediatric solid cancers fail to respond to standard immuno-oncology agents relying on preexisting adaptive antitumor immune responses. The adoptive transfer of tumor-antigen specific T cells, such as CAR-gene modified T cells, is an attractive strategy, but its efficacy has been limited. Evidence is accumulating that local barriers in the tumor microenvironment prevent the infiltration of T cells and impede therapeutic immune responses. A thorough understanding of the components of the functional compartment of the tumor microenvironment and their interaction could inform effective combination therapies and novel engineered therapeutics, driving immunotherapy towards its full potential in pediatric patients. This review summarizes current knowledge on the cellular composition and significance of the tumor microenvironment in common extracranial solid cancers of childhood and adolescence, such as embryonal tumors and bone and soft tissue sarcomas, with a focus on myeloid cell populations that are often present in abundance in these tumors. Strategies to (co)target immunosuppressive myeloid cell populations with pharmacological anticancer agents and with selective antagonists are presented, as well as novel concepts aiming to employ myeloid cells to cooperate with antitumor T cell responses.

Simple Summary GD2 is an antigen that is tumor-specific and can be used as a target for specific immunotherapies. Since the knowledge about GD2 in breast cancer is limited, we analyzed the frequency of GD2 expression in breast cancer using two different staining methods and the impact of GD2 expression on the survival of breast cancer patients. GD2 expression was found in more than 50% of breast cancer cases, with the highest frequency in hormone receptor-positive tumors. GD2 expression was not significantly associated with patient outcome. Unlike previous studies with smaller sample sizes that lacked correlation with clinical data, this study includes a larger cohort and associations with survival data and shows that GD2 is expressed on human breast cancer cells, providing a potential target for immunotherapies (e.g., anti-GD2 antibodies or GD2 CAR T cells), that are currently undergoing clinical testing. Abstract The disialoganglioside GD2 is a tumor-associated antigen that may allow for the application of targeted immunotherapies (anti-GD2 antibodies, GD2 CAR T cells) in patients with neuroblastoma and other solid tumors. We retrospectively investigated GD2 expression in a breast cancer cohort, using immunohistochemistry (IHC) and immunofluorescence (IF) on tissue microarrays (TMAs), and its impact on survival. GD2 expression on IHC (n = 568) and IF (n = 503) was investigated in relation to subtypes and patient outcome. Overall, 50.2% of the 568 IHC-assessed samples and 69.8% of the 503 IF-assessed samples were GD2-positive. The highest proportion of GD2-positive tumors was observed in luminal tumors. Significantly fewer GD2-positive cases were detected in triple-negative breast cancer (TNBC) compared with other subtypes. The proportion of GD2-expressing tumors were significantly lower in HER2-positive breast cancer in comparison with luminal tumors on IF staining (but not IHC). GD2 expression of IHC or IF was not significantly associated with disease-free or overall survival, in either the overall cohort or in individual subtypes. However, GD2 expression can be seen in more than 50% of breast cancer cases, with the highest frequency in hormone receptor-positive tumors. With this high expression frequency, patients with GD2-positive advanced breast cancer of all subtypes may benefit from GD2-targeting immunotherapies, which are currently subject to clinical testing.

// Christian Spurny 1 , Sareetha Kailayangiri 1 , Bianca Altvater 1 , Silke Jamitzky 1 , Wolfgang Hartmann 2 , Eva Wardelmann 2 , Andreas Ranft 1, 3 , Uta Dirksen 1, 3 , Susanne Amler 4 , Jendrik Hardes 5, 4 , Maike Fluegge 1 , Jutta Meltzer 1 , Nicole Farwick 1 , Lea Greune 1 and Claudia Rossig 1, 6 1 Department of Pediatric Hematology and Oncology, University Children´s Hospital Muenster, Muenster, Germany 2 Gerhard Domagk Institute of Pathology, University of Muenster, Muenster, Germany 3 University Hospital Essen, Pediatrics III, West German Cancer Centre, Essen, Germany 4 Institute of Biostatistics and Clinical Research, University of Muenster, Muenster, Germany 5 Department of Orthopedic Surgery, University Hospital Muenster, Muenster, Germany 6 Cells-in-Motion Cluster of Excellence (EXC 1003 – CiM), University of Muenster, Germany Correspondence to: Claudia Rossig, email: rossig@uni-muenster.de Keywords: Ewing sarcoma; HLA-G; T cells; cellular immunotherapy; immune checkpoints Received: September 08, 2017 Accepted: October 27, 2017 Published: December 22, 2017 ABSTRACT Ewing sarcoma (EwS) is an aggressive mesenchymal cancer of bones or soft tissues. The mechanisms by which this cancer interacts with the host immune system to induce tolerance are not well understood. We hypothesized that the non-classical, immune-inhibitory HLA-molecule HLA-G contributes to immune escape of EwS. While HLA-G pos suppressor T cells were not increased in the peripheral blood of EwS patients, HLA-G was locally expressed on the tumor cells and/or on infiltrating lymphocytes in 16 of 47 pretherapeutic tumor biopsies and in 4 of 12 relapse tumors. HLA-G expression was not associated with risk-related patient variables or response to standard chemotherapy, but with significantly increased numbers of tumor-infiltrating CD3+ T cells compared to HLA-G neg EwS biopsies. In a mouse model, EwS xenografts after adoptive therapy with tumor antigen-specific CAR T cells strongly expressed HLA-G whereas untreated control tumors were HLA-G neg . IFN-γ stimulation of EwS cell lines in vitro induced expression of HLA-G protein. We conclude that EwS cells respond to tumor-infiltrating T cells by upregulation of HLA-G, a candidate mediator of local immune escape. Strategies that modulate HLA-G expression in the tumor microenvironment may enhance the efficacy of cellular immunotherapeutics in this cancer.

Background T cells engineered to express chimeric antigen receptors (CARs) are a novel modality to treat refractory cancers. The development of CAR T cells against Ewing sarcoma (EwS) is limited by a lack of targetable surface antigens. We investigated vascular endothelial growth factor receptor 2 (VEGFR2) expressed on tumor-associated blood vessels as potential CAR target in this cancer. Methods Expression of VEGFR2 was studied by immunohistochemistry in human EwS biopsies and in murine xenografts and by flow cytometry in EwS cell lines. CARs with short, medium, and long hinge domains against either human or murine VEGFR2 were generated and expressed in human T cells by retroviral gene transfer. The capacity of the individual CARs to activate T cells in response to VEGFR2-expressing cells was compared in vitro. Results Tumor-associated endothelial cells in human EwS biopsies and in xenografts expressed VEGFR2. Tumor cells in the majority of EwS biopsies were also VEGFR2-positive. Following modification with anti-mouse or anti-human VEGFR2-specific CAR genes, T cells specifically lysed VEGFR2-expressing target cells of the respective species. CAR T cells with short-length or medium-length hinge domains were functionally superior over those with the long hinge region by in vitro parameters, including antigen-specific degranulation responses, lysis of tumor spheroids, tumor necrosis factor α secretion, sequential killing, and proliferation. Conclusions VEGFR2 is consistently expressed on endothelial cells of the tumor stroma in EwS and thus is a candidate target for CAR T cells in this cancer. Among various VEGFR2-specific CARs, a construct with a short hinge domain was chosen to be further developed toward clinical translation.

Chimeric antigen receptor (CAR) engineering of T cells allows one to specifically target tumor cells via cell surface antigens. A candidate target in Ewing sarcoma is the ganglioside G(D2), but heterogeneic expression limits its value. Here we report that pharmacological inhibition of Enhancer of Zeste Homolog 2 (EZH2) at doses reducing H3K27 trimethylation, but not cell viability, selectively and reversibly induces G(D2) surface expression in Ewing sarcoma cells. EZH2 in Ewing sarcoma cells directly binds to the promoter regions of genes encoding for two key enzymes of G(D2) biosynthesis, and EZH2 inhibition enhances expression of these genes. G(D2) surface expression in Ewing sarcoma cells is not associated with distinct in vitro proliferation, colony formation, chemosensitivity, or in vivo tumorigenicity. Moreover, disruption of G(D2) synthesis by gene editing does not affect its in vitro behavior. EZH2 inhibitor treatment sensitizes Ewing sarcoma cells to effective cytolysis by G(D2)-specific CAR gene-modified T cells. In conclusion, we report a clinically applicable pharmacological approach for enhancing efficacy of adoptively transferred G(D2)-redirected T cells against Ewing sarcoma, by enabling recognition of tumor cells with low or negative target expression.

PD-1 checkpoint blockade in patients with relapsed AML after allogeneic stem cell transplantation

Preclinical in vivo studies of chimeric antigen receptor (CAR) T cells often rely on NOD-scid gamma (NSG) mouse models which lack T cells, B cells and NK cells and thereby allow for reliable engraftment of human tumor xenografts. But murine myeloid cells present in the NSG mouse strain can affect the tumor microenvironment as well as the function of adoptively transferred human immune effector cells. Long et al. (Cancer Immunol Res. 2016 Oct;4(10):869-880) reported that pediatric sarcoma xenografts in NSG mice induce in vivo expansion of murine CD11b+ myeloid-derived suppressor cells (MDSC), and that this cell population suppresses human CAR T cell proliferation in vitro. We investigated a strategy to avoid inhibitory effects of murine MDSCs on Ewing sarcoma xenografts in NSG mice by in vivo pretreatment with murine antibody against two cell surface antigens, Ly6C/Ly-6G (granulocyte-differentiation antigen-1, Gr-1), expressed on murine MDSC. Analysis of the CD11b+ myeloid cell populations in NSG mice 17-37 days after subcutaneous transplantation of the Ewing sarcoma cell line TC-71 showed a noticeable increase of CD11b+ cells in the peripheral blood compared to non-tumor bearing NSG mice (median 3.4 × 103, range 0.7-15.5 × 103 cells/ml, n=8 vs median 2.1 × 103, range 1.2-4.0 × 103 cells/ml, n=7). The increase was even more pronounced in the spleens, with a median absolute number of 2.6 × 106 CD11b+ cells (range 0.2-21 × 106 cells) per spleen in sarcoma-bearing mice versus 0.5 × 106 CD11b+ cells (range 0.2-0.8 × 106 cells) per spleen in mice without tumors. The granulocytic MDSC subset coexpressing Ly6Gpos was the most prominent subpopulation in spleen and blood. To eliminate murine MDSCs, we treated sarcoma-bearing mice twice weekly with 200 µg anti-Gr1 antibody RB6-8C5 over a period of 2 weeks, starting at tumor volumes of 100-200 mm3. While the combined percentage of the Ly6Gpos and Ly6Cpos cell populations in the spleens decreased compared to untreated mice (median 55%, range 36-75%, n=9 versus median 74%, range 72-86%, n=8), the total numbers of CD11b+ cells further increased (median 3.1 × 106, range 0.4 × 106-11 × 106 cells/spleen). Our data suggest that anti-Gr1 antibody pretreatment leads to blockade of the Ly6C/Ly6G receptors rather than eliminating MDSC subsets. To optimize preclinical pediatric sarcoma models, other methods for depletion or functional inactivation of mouse MDSCs will need to be evaluated. Citation Format: Sareetha Kailayangiri, Bianca Altvater, Katja Urban, Jutta Meltzer, Lea Greune, Nicole Farwick, Silke Jamitzky, Claudia Rossig. Evaluation of anti-Gr1 antibody for depletion of MDSC in preclinical NSG mouse models of pediatric sarcoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4999.

Chimeric antigen receptor (CAR) gene-modified T cells (CAR T cells) can eradicate B cell malignancies via recognition of surface-expressed B lineage antigens. Antigen escape remains a major mechanism of relapse and is a key barrier for expanding the use of CAR T cells towards solid cancers with their more diverse surface antigen repertoires. In this review we discuss strategies by which cancers become amenable to effective CAR T cell therapy despite heterogeneous phenotypes. Pharmaceutical approaches have been reported that selectively upregulate individual target antigens on the cancer cell surface to sensitize antigen-negative subclones for recognition by CARs. In addition, advanced T cell engineering strategies now enable CAR T cells to interact with more than a single antigen simultaneously. Still, the choice of adequate targets reliably and selectively expressed on the cell surface of tumor cells but not normal cells, ideally by driving tumor growth, is limited, and even dual or triple antigen targeting is unlikely to cure most solid tumors. Innovative receptor designs and combination strategies now aim to recruit bystander cells and alternative cytolytic mechanisms that broaden the activity of CAR-engineered T cells beyond CAR antigen-dependent tumor cell recognition.

Background Patients with primary metastatic or relapsed Ewing sarcomas (EwS) have a poor prognosis. While inhibitory insulin-like growth factor 1 receptor (IGF-1R)-specific antibodies have shown single agent activity in some patients with refractory disease, effective therapeutic targeting will rely on optimal combinations with conventional or innovative therapies. Specifically, combination of inhibitory IGF-1R antibodies with adoptive transfer of activated natural killer (NK) cells may have therapeutic benefit in EwS without adding toxicity. Procedure We investigated the in vitro effects of IGF-1R targeting on the immunological profile of EwS cells and on the survival and tumor targeting capacity of K-562-activated NK cells. Results IGF-1R inhibition reliably reduced EwS cell viability without affecting expression of immune-modulatory and MHC molecules. In NK cells, we observed a significant superior expansion following in vitro activation in the presence of IGF-1R-specific antibodies, while expression of differentiation markers and activating receptors remained unaffected. Activated NK cells coincubated with EwS cells showed potent degranulation responses unaffected by IGF-1R inhibition. These findings were reproducible in a stimulator cell-free NK cell expansion system, suggesting that direct effects of IGF-R1 antibodies on the IGF-R1 pathway in NK cells induce their activation and expansion. Conclusions Activated human NK cells respond to IGF-1R inhibition with superior expansion kinetics while maintaining potent antitumor responses against EwS. Combination of adoptive NK cell transfer with IGF-1R targeting may be an efficient means to eliminate minimal residual disease after conventional therapy and thereby rescue patients at the highest risk of relapse. Pediatr Blood Cancer © 2015 Wiley Periodicals, Inc.

Background Programmed cell death 1 (PD-1) receptor engagement on T cells by its ligand programmed cell death ligand 1 (PD-L1) is a key mechanism of immune escape, and antibody blockade of the interaction has emerged as an effective immunotherapeutic strategy in some cancers. The role and relevance of the PD-1 checkpoint in Ewing sarcoma (EwS) is not yet understood. Procedure Here, we investigated expression of PD-L1 and PD-1 in EwS by immunohistochemistry analysis of pretherapeutic tumor biopsies and in tumor xenografts following treatment with human T cells engineered to express a chimeric antigen receptor (CAR) against the tumor-associated antigen GD2. PD-L1 surface expression in EwS cell lines was assessed by flow cytometry. Results PD-L1 expression was not detectable on tumor cells in any of the 60 EwS biopsies. Infiltrating PD-L1 positive T cells were found in one tumor, and four biopsies contained PD-1-positive T cells. Of 13 EwS cell lines, none constitutively expressed PD-L1 on the cell surface. Interferon-γ cytokine stimulation induced upregulation of the ligand on all cell lines. Adoptive therapy with CAR gene-modified T cells in a mouse model did not induce PD-L1 expression in EwS xenografts despite tumor infiltration with PD-1+ CD3+ T cells. Conclusions EwS cells can upregulate PD-L1 under inflammatory conditions, but do not express the ligand in the pretherapeutic tumor microenvironment or postexposure to CAR T cells. PD-1 checkpoint blockade alone is thus unlikely to evoke potent immune responses against EwS. Identification of the relevant immune evasion strategies in EwS will be vital for the development of effective immune targeting strategies.

Disseminated or relapsed Ewing sarcoma (EwS) has remained fatal in the majority of patients. A promising approach to preventing relapse after conventional therapy is to establish tumor antigen-specific immune control. Efficient and specific T cell memory against the tumor depends on the expansion of rare T cells with native specificity against target antigens overexpressed by the tumor. Candidate antigens in EwS include six-transmembrane epithelial antigen of the prostate-1 (STEAP1), and the human cancer/testis antigens X-antigen family member 1 (XAGE1) and preferentially expressed antigen in melanoma (PRAME). Here, we screened normal donors and EwS patients for the presence of circulating T cells reactive with overlapping peptide libraries of these antigens by IFN-γ Elispot analysis. The majority of 22 healthy donors lacked detectable memory T cell responses against STEAP1, XAGE1 and PRAME. Moreover, ex vivo detection of T cells specific for these antigens in both blood and bone marrow were limited to a minority of EwS patients and required nonspecific T cell prestimulation. Cytotoxic T cells specific for the tumor-associated antigens were efficiently and reliably generated by in vitro priming using professional antigen-presenting cells and optimized cytokine stimulation; however, these T cells failed to interact with native antigen processed by target cells and with EwS cells expressing the antigen. We conclude that EwS-associated antigens fail to induce efficient T cell receptor (TCR)-mediated antitumor immune responses even under optimized conditions. Strategies based on TCR engineering could provide a more effective means to manipulating T cell immunity toward targeted elimination of tumor cells.

Introduction: Synovial sarcoma (SySa) is a rare soft-tissue malignancy characterized by a specific reciprocal translocation t(X;18). The resulting chimeric SS18-SSX fusion protein acts as transcriptional dysregulator representing the major oncogenic driver in SySa tumorigenesis. Since targeting the fusion protein itself remains a particular challenge, it appears reasonable to therapeutically address signaling pathways which are functionally dependent on the SS18-SSX fusion protein. As different tumor entities were recently shown to harbor aberrant Hippo signaling patterns leading to increased activity of the transcriptional coactivators YAP/TAZ, the aim of this study was to analyze the role of YAP/TAZ in SySa and to decipher the functional link to the SS18-SSX fusion protein. Experimental procedures: YAP/TAZ expression was analyzed by immunohistochemistry in a large cohort of SySa tissue specimens (n=65). Five SySa cell lines and mesenchymal SCP-1 stem cells stably expressing the SS18-SSX fusion protein were employed for in vitro analyses. We set out to analyze whether YAP/TAZ-TEAD transcriptional activity is dependent on the SySa-specific fusion protein, if this dependency is mediated by IGF-IR signaling (known to be activated in SySa) and to understand the biological function of YAP/TAZ in SySa. To modulate YAP/TAZ-TEAD transcriptional activity, RNAi-mediated knockdown and the small molecule inhibitor verteporfin were applied. Finally, the therapeutic effect of YAP/TAZ inhibition was tested in vivo using SySa cell line-based and patient-derived xenografts. Results: SySa tissue specimens and cell lines strongly expressed nuclear YAP/TAZ. RNAi-mediated knockdown of SS18-SSX fusion protein led to significant reduction of YAP/TAZ-TEAD transcriptional activity while SS18-SSX overexpression in SCP-1 cells induced aberrant YAP/TAZ signals. This regulatory connection was at least partly realized through an IGF-II/IGF-IR loop, in which the SS18-SSX fusion protein drives IGF2 expression causing dysregulation of the Hippo effectors LATS1 and MOB1, eventually leading to YAP/TAZ activation. Inhibition of YAP/TAZ-TEAD transcriptional activity by RNAi or verteporfin resulted in a significant induction of apoptosis and significant reduction of SySa cell growth in vitro and in vivo. Conclusions: Our study reveals SS18-SSX fusion protein-driven YAP/TAZ-TEAD signals to play an elementary role in SySa. Given the high efficacy of YAP/TAZ-directed pharmacological approaches in SySa xenografts, this preclinical study may constitute the basis for a novel therapeutic strategy to inhibit SS18-SSX-driven tumorigenesis. Citation Format: Ilka Isfort, Magdalene Cyra, Sandra Elges, Sareetha Kailayangiri, Bianca Altvater, Claudia Rossig, Jan-Henrik Mikesch, Agnieszka Wozniak, Patrick Schöffski, Eva Wardelmann, Marcel Trautmann, Wolfgang Hartmann. SS18-SSX modulates YAP/TAZ-TEAD transcriptional activity in synovial sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 388.

Background: Novel treatment strategies in Ewing sarcoma include targeted cellular therapies. Preclinical in vivo models are needed that reflect their activity against systemic (micro)metastatic disease. Methods: Whole-body magnetic resonance imaging (WB-MRI) was used to monitor the engraftment and dissemination of human Ewing sarcoma xenografts in mice. In this model, we evaluated the therapeutic efficacy of T cells redirected against the Ewing sarcoma-associated antigen GD2 by chimeric receptor engineering. Results: Of 18 mice receiving intravenous injections of VH-64 Ewing sarcoma cells, all developed disseminated tumour growth detectable by WB-MRI. All mice had lung tumours, and the majority had additional manifestations in the bone, soft tissues, and/or kidney. Sequential scans revealed in vivo growth of tumours. Diffusion-weighted whole-body imaging with background signal suppression effectively visualised Ewing sarcoma growth in extrapulmonary sites. Animals receiving GD2-targeted T-cell therapy had lower numbers of pulmonary tumours than controls, and the median volume of soft tissue tumours at first detection was lower, with a tumour growth delay over time. Conclusion: Magnetic resonance imaging reliably visualises disseminated Ewing sarcoma growth in mice. GD2-retargeted T cells can noticeably delay tumour growth and reduce pulmonary Ewing sarcoma manifestations in this aggressive disease model.

Trials with second generation CD19 chimeric antigen receptors (CAR) T-cells report unprecedented responses but are associated with risk of cytokine release syndrome (CRS). Instead, we studied the use of donor Epstein–Barr virus-specific T-cells (EBV CTL) transduced with a first generation CD19CAR, relying on the endogenous T-cell receptor for proliferation. We conducted a multi-center phase I/II study of donor CD19CAR transduced EBV CTL in pediatric acute lymphoblastic leukaemia (ALL). Patients were eligible pre-emptively if they developed molecular relapse (>5 × 10-4) post first stem cell transplant (SCT), or prophylactically post second SCT. An initial cohort showed poor expansion/persistence. We therefore investigated EBV-directed vaccination to enhance expansion/persistence. Eleven patients were treated. No CRS, neurotoxicity or graft versus host disease (GVHD) was observed. At 1 month, 5 patients were in CR (4 continuing, 1 de novo), 1 PR, 3 had stable disease and 3 no response. At a median follow-up of 12 months, 10 of 11 have relapsed, 2 are alive with disease and 1 alive in CR 3 years. Although CD19CAR CTL expansion was poor, persistence was enhanced by vaccination. Median persistence was 0 (range: 0–28) days without vaccination compared to 56 (range: 0–221) days with vaccination (P=0.06). This study demonstrates the feasibility of multi-center studies of CAR T cell therapy and the potential for enhancing persistence with vaccination.Leukemia advance online publication, 10 March 2017; doi:10.1038/leu.2017.39

Activated and in vitro expanded natural killer (NK) cells have substantial cytotoxicity against many tumor cells, but their in vivo efficacy to eliminate solid cancers is limited. Here, we used chimeric antigen receptors (CARs) to enhance the activity of NK cells against Ewing sarcomas (EwS) in a tumor antigen-specific manner. Expression of CARs directed against the ganglioside antigen GD2 in activated NK cells increased their responses to GD2+ allogeneic EwS cells in vitro and overcame resistance of individual cell lines to NK cell lysis. Second-generation CARs with 4-1BB and 2B4 co-stimulatory signaling and third-generation CARs combining both co-stimulatory domains were all equally effective. By contrast, adoptive transfer of GD2-specific CAR gene-modified NK cells both by intratumoral and intraperitoneal delivery failed to eliminate GD2-expressing EwS xenografts. Histopathology review revealed upregulation of the immunosuppressive ligand HLA-G in tumor autopsies from mice treated with NK cells compared to untreated control mice. Supporting the relevance of this finding, in vitro co-incubation of NK cells with allogeneic EwS cells induced upregulation of the HLA-G receptor CD85j, and HLA-G1 expressed by EwS cells suppressed the activity of NK cells from three of five allogeneic donors against the tumor cells in vitro. We conclude that HLA-G is a candidate immune checkpoint in EwS where it can contribute to resistance to NK cell therapy. HLA-G deserves evaluation as a potential target for more effective immunotherapeutic combination regimens in this and other cancers.

Background: Novel treatment strategies are needed to cure disseminated Ewing sarcoma. Primitive neuroectodermal features and a mesenchymal stem cell origin are both compatible with aberrant expression of the ganglioside antigen GD2 and led us to explore GD2 immune targeting in this cancer. Methods: We investigated GD2 expression in Ewing sarcoma by immunofluorescence staining. We then assessed the antitumour activity of T cells expressing a chimeric antigen receptor specific for GD2 against Ewing sarcoma in vitro and in vivo. Results: Surface GD2 was detected in 10 out of 10 Ewing sarcoma cell lines and 3 out of 3 primary cell cultures. Moreover, diagnostic biopsies from 12 of 14 patients had uniform GD2 expression. T cells specifically modified to express the GD2-specific chimeric receptor 14. G2a-28ζ efficiently interacted with Ewing sarcoma cells, resulting in antigen-specific secretion of cytokines. Moreover, chimeric receptor gene-modified T cells from healthy donors and from a patient exerted potent, GD2-specific cytolytic responses to allogeneic and autologous Ewing sarcoma, including tumour cells grown as multicellular, anchorage-independent spheres. GD2-specific T cells further had activity against Ewing sarcoma xenografts. Conclusion: GD2 surface expression is a characteristic of Ewing sarcomas and provides a suitable target antigen for immunotherapeutic strategies to eradicate micrometastatic cells and prevent relapse in high-risk disease.

Specific cellular immunotherapy of cancer requires efficient generation and expansion of cytotoxic T lymphocytes (CTLs) that recognize tumor-associated self-antigens. Here, we investigated the capacity of human γδ T cells to induce expansion of CD8+ T cells specific for peptides derived from the weakly immunogenic tumor-associated self-antigens PRAME and STEAP1. Coincubation of aminobisphosphonate-stimulated human peripheral blood-derived γδ T cells (Vγ9+Vδ2+), loaded with HLA-A*02-restricted epitopes of PRAME, with autologous peripheral blood CD8+ T cells stimulated the expansion of peptide-specific cytolytic effector memory T cells. Moreover, peptide-loaded γδ T cells efficiently primed antigen-naive CD45RA+ CD8+ T cells against PRAME peptides. Direct comparisons with mature DCs revealed equal potency of γδ T cells and DCs in inducing primary T-cell responses and peptide-specific T-cell activation and expansion. Antigen presentation by γδ T-APCs was not able to overcome the limited capacity of peptide-specific T cells to interact with targets expressing full-length antigen. Importantly, T cells with regulatory phenotype (CD4+ CD25hiFoxP3+) were lower in cocultures with γδ T cells compared to DCs. In summary, bisphosphonate-activated γδ T cells permit generation of CTLs specific for weakly immunogenic tumor-associated epitopes. Exploiting this strategy for effective immunotherapy of cancer requires strategies that enhance the avidity of CTL responses to allow for efficient targeting of cancer.

Safe and effective targeting of cancer with chimeric antigen receptor (CAR) engineered T cells relies on the presence of adequate tumor-associated surface antigens. We have found that Ewing sarcomas (EwS) express ganglioside GD2 and are recognized by T cells engineered with GD2-specific CARs. In an extended series of 97 EwS patient samples, 52 were GD2pos by immunohistochemistry. Based on reports that GD2 in breast cancer defines a malignant population with stem cell characteristics, we hypothesized that GD2 expression in EwS is associated with high capacity to self-renew and reinitiate tumor growth. Among 15 individual EwS cell lines with variable surface expression of GD2 by flow cytometry, GD2 expression levels were not associated with the capacities to proliferate and expand in vitro, form colonies in semi-solid medium, nor with their chemosensitivity, assessed by cell viabilities in the presence of increasing concentrations of the cytotoxic drug doxorubicin. Subpopulations with GD2hi phenotype selected from GD2pos and from GD2low EwS cell lines by cell sorting maintained their GD2hi and GD2low expression status during subsequent cultures over several weeks. The two subpopulations did not have different in vitro growth, colony-formation capacity, or chemosensitivity. In xenografting experiments, GD2hi and GD2low subpopulations of two EwS cell lines initiated tumors with comparable efficacies. To obtain direct evidence that GD2 surface expression is irrelevant for the biology of EwS cells, we performed genetic knockdown of the GD3S gene which drives biosynthesis of GD2 by CRISPR/Cas9 gene editing. GD3S gene editing resulted in effective elimination of GD2 surface expression in the GD2hi EwS cell lines TC-71, VH-64 and A4573. The knockdown did not affect the capacity of the cells to proliferate, form colonies in soft agar in vitro, nor their chemosensitivity compared to wild-type EwS cells from the individual cell lines. We conclude that GD2 expression in EwS cells, other than reported in breast cancer, is not associated with distinct functional features. Specifically, GD2 does not affect the growth characteristics, clono- and tumorigenicity and chemosensitivity of the tumor cells. Elimination of GD2pos subpopulations from heterogeneous tumors by CAR T cell targeting is therefore unlikely to eradicate the disease and will have to be combined with additional targeting strategies. Citation Format: Sareetha Kailayangiri, Bianca Altvater, Silke Jamitzky, Stefanie Lesch, Jan-Henrik Mikesch, Sonja Schelhaas, Schaefers Michael, Jutta Meltzer, Nicole Farwick, Wolfgang Hartmann, Eva Wardelmann, Petra Fischer, Jendrik Hardes, Claudia Rossig. Expression of ganglioside GD2 in Ewing sarcoma cells is not associated with specific functional characteristics or stem cell features [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4631.

Purpose: Novel natural killer (NK) cell–directed strategies in cancer immunotherapy aim at specifically modulating the balance between NK cell receptor signals toward tumor-specific activation. The signaling lymphocyte activation molecule–related receptor 2B4 (CD244) is an important regulator of NK cell activation. We investigated whether 2B4-enhanced activation signals can redirect the cytolytic function of human NK cells to NK cell–resistant and autologous leukemia and tumor targets.Experimental Design: In vitro–stimulated NK cells from healthy donors and pediatric leukemia patients were gene modified with CD19 or GD2-specific chimeric receptors containing either the T-cell receptor ζ or 2B4 endodomain alone or combined.Results: Chimeric 2B4 signaling alone failed to induce interleukin-2 receptor up-regulation and cytokine secretion but triggered a specific degranulation response. Integration of the 2B4 endodomain into T-cell receptor ζ chimeric receptors significantly enhanced all aspects of the NK cell activation response to antigen-expressing leukemia or neuroblastoma cells, including CD25 up-regulation, secretion of IFN-γ and tumor necrosis factor-α, release of cytolytic granules, and growth inhibition, and overcame NK cell resistance of autologous leukemia cells while maintaining antigen specificity.Conclusion: These data indicate that the 2B4 receptor has a potent costimulatory effect in NK cells. Antigen-specific 2B4ζ-expressing NK cells may be a powerful new tool for adoptive immunotherapy of leukemia and other malignancies.

Patients with primary metastatic or relapsed Ewing sarcomas (EwS) have a poor prognosis. While inhibitory insulin-like growth factor 1 receptor (IGF-1R)-specific antibodies have shown single agent activity in some patients with refractory disease, effective therapeutic targeting will rely on optimal combinations with conventional or innovative therapies. Specifically, combination of inhibitory IGF-1R antibodies with adoptive transfer of activated natural killer (NK) cells may have therapeutic benefit in EwS without adding toxicity.We investigated the in vitro effects of IGF-1R targeting on the immunological profile of EwS cells and on the survival and tumor targeting capacity of K-562-activated NK cells.IGF-1R inhibition reliably reduced EwS cell viability without affecting expression of immune-modulatory and MHC molecules. In NK cells, we observed a significant superior expansion following in vitro activation in the presence of IGF-1R-specific antibodies, while expression of differentiation markers and activating receptors remained unaffected. Activated NK cells coincubated with EwS cells showed potent degranulation responses unaffected by IGF-1R inhibition. These findings were reproducible in a stimulator cell-free NK cell expansion system, suggesting that direct effects of IGF-R1 antibodies on the IGF-R1 pathway in NK cells induce their activation and expansion.Activated human NK cells respond to IGF-1R inhibition with superior expansion kinetics while maintaining potent antitumor responses against EwS. Combination of adoptive NK cell transfer with IGF-1R targeting may be an efficient means to eliminate minimal residual disease after conventional therapy and thereby rescue patients at the highest risk of relapse.

Novel treatment strategies for Ewing sarcoma aim to eliminate residual tumor cells that have maintained the capacity to reinitiate tumor growth after intensive conventional therapy. Preclinical models that more closely mimic in vivo tumor growth than standard monolayer cultures are needed. Sphere formation under anchorage-independent, serum-free conditions has been proposed to enrich for cells with tumor-initiating, stem cell-like properties in various solid cancers. In the present study, we assessed the phenotype and functional stem cell characteristics of Ewing sarcoma spheres. Spheres were generated under serum-free culture conditions from four Ewing sarcoma cell lines and four relapse tumor biopsies. Standard monolayer cultures were established as controls. Median levels of surface expression of the Ewing sarcoma marker CD99 as well as the supposed stem cell marker CD133 and the neural crest marker CD57 were comparable between spheres and monolayers. Ewing sarcoma spheres from individual tumors failed to continuously self-renew by secondary sphere formation. They contained variable proportions of side populations (SPs). Sphere culture did not enhance the in vivo tumorigenicity of Ewing sarcoma cells in a murine xenograft model. We conclude that sphere formation under serum-free conditions is not a reliable tool to enrich for cells with stem cell characteristics in Ewing sarcoma. By mimicking the anchorage-independent, multicellular growth of Ewing sarcoma micrometastases, in vitro sphere growth may still add value as a preclinical tool to evaluate the efficacy of novel therapeutics.

Ufm1 (ubiquitin-fold modifier 1) is the most recently identified member of the ubiquitin-like protein family. We characterized the Ufm1 cascade of the model organism Caenorhabditis elegans in terms of function and analyzed interactions of the involved proteins in vitro and in vivo. Furthermore, we investigated the phenotypes of the deletion mutants uba5(ok3364) (activating enzyme of Ufm1) and ufc1(tm4888) (conjugating enzyme of Ufm1). The viable deletion mutants showed a decrease in reproduction, development, life span, and a reduced survival under heavy metal stress. However, an increased survival rate under pathogenic, oxidative, heat, and endoplasmic reticulum stress was observed. We propose that the Ufm1 cascade negatively regulates the IRE1-mediated unfolded protein response.

The in vivo persistence of chimeric antigen receptor (CAR) modified T cells is a major prerequisite for their antitumor activity and was found to be associated with a less differentiated immune phenotype. Here, we compared two in vitro T cell stimulation conditions: coated anti-CD3/CD28 antibodies [3/28], and Dynabead stimulation of enriched CD3+ T cells [DB]. Peripheral blood T cells from three healthy donors were stimulated with either of the two methods, retrovirally transduced with the GD2-specific CAR GD2-BBz on day 2 or 3, and expanded in RPMI/AIMV medium with 50 IU/ml recombinant human interleukin-2 for 13 days. T cell expansion rates were comparable between the two stimulation conditions and independent of CAR gene expression. Transduction efficiencies, determined by staining with the GD2-CAR-specific antibody Ganglidiomab, were also comparable. The immune phenotype by expression of CD3, CD4, CD8, CD45RO and CD197 was determined by flow cytometry analysis on day 13 or 14 after initial stimulation. The proportions of central memory (TCM), effector memory (TEM) or naïve T cells (TN) within the two types of cultures were noticeably different (Table 1), with a higher proportion of non-transduced CD8+ T cells with a TCM phenotype after DB compared to CD3/CD28 stimulation (p = 0.005 for DB d2, p = 0.01 for DB d3). Compared to non-transduced T cells, CAR-expressing cells of all types of cultures had higher proportions of TCM cells (p = 0.02 for CD4+ T cells, p Citation Format: Laurin Ochs, Bianca Altvater, Sareetha Kailayangiri, Christian Spurny, Claudia Rossig, Silke Jamitzky. Different stimulation conditions affect the immune phenotype of GD2-specific chimeric antigen receptor-expressing T cells. [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 2314.

Ewing Sarcoma (EwS) is an aggressive malignancy of bone and soft tissue which still lacks efficient treatment in case of metastases and relapse. Cellular immunotherapies for EwS are under development, but inhibitory molecules in the tumor microenvironment may counteract antitumor immune responses by preexisting or therapeutic immune effector cells. Here we hypothesized that the non-classical HLA-molecule HLA-G may contribute to immune escape of EwS. HLA-G is a potent inhibitor of both T cells and NK cells. It is naturally expressed on trophoblast cells during pregnancy as well as on mesenchymal stem cells from which EwS cells are thought to originate. We analyzed expression of membrane-bound HLA-G1 by flow cytometry and expression of shedded HLA-G1 and soluble HLA-G5 by ELISA in 14 EwS cell lines with and without stimulation with interferon-γ (IFN-γ). Whereas all cell lines failed to express HLA-G1 both before and after IFN-γ stimulation, and none secreted HLA-G without stimulation, 1 of 14 cell lines (TC-32) responded to IFN-γ stimulation by significant upregulation of soluble HLA-G (p = 0.004). To study HLA-G expression in EwS within their tumor microenvironment, we analyzed paraffin-embedded pretherapeutic tumor biopsies from 35 patients by IHC using the HLA-G specific antibody clone 4H84 and detected HLA-G expression in 12 cases (34%), either on the tumor cells (10/35) and/or on infiltrating lymphocytes (7/35). We further studied the presence of soluble HLA-G and HLA-G+ T cells in the peripheral blood of 19 EwS patients and 15 healthy donors. Serum HLA-G was not increased in the EwS patients compared to healthy controls. Moreover, no significant difference in the proportions of naturally occurring HLA-G+CD4+ (Mean 0.9±0.8% vs. 0.9±0.6%, p = 0.627) or HLA-G+CD8+ (Mean 1.2±1.2% vs. 1.7±1.0%, p = 0.134) suppressor T cells among peripheral blood lymphocytes was found between EwS patients and healthy donors by flow cytometry. Thus, systemic HLA-G secretion and expression is unlikely to have a major role in EwS, but the presence of HLA-G+ cells found in EwS biopsies in a substantial proportion of patients deserves further exploration. To address the potential functional relevance of HLA-G+ cells in the tumor microenvironment, we expressed HLA-G1 in 2 EwS cell lines by retroviral gene transfer. Coincubation of HLA-G-expressing EwS cells with freshly isolated allogeneic NK-cells resulted in suppression of EwS cell lysis by NK cells in 3 of 6 NK cell donors in a flow cytometry based cytotoxicity assay. In detail, HLA-G+ EwS cells suppressed NK-cell cytotoxicity up to 47.0±14.8%, (VH-64, p = 0.001) and up to 87.0±16.0% (WE-68, p = 0.002) compared to mock transduced control. We conclude that local expression of HLA-G within the tumor microenvironment in EwS is a candidate mediator of immune escape and a potential barrier to cellular immunotherapeutics. Strategies that modulate HLA-G expression may be effective to overcome local immune suppression in this cancer. Citation Format: Christian Spurny, Bianca Altvater, Sareetha Kailayangiri, Silke Landmeier, Martina Ahlmann, Uta Dirksen, Andreas Ranft, Heinz Wiendl, Wolfgang Hartmann, Eva Wardelmann, Claudia Rossig. Immune-inhibitory HLA-G is expressed in the tumor microenvironment of Ewing Sarcomas. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 458. doi:10.1158/1538-7445.AM2015-458

Despite optimization of modern treatment strategies, patients with primary metastatic Ewing sarcomas or with relapsed disease have a poor prognosis. The insulin-like growth factor 1 receptor (IGF-1R) pathway is a target of the disease-defining translocations and important for the biology of Ewing sarcomas. IGF-1R antagonists have shown activity in some patients with refractory disease. More effective therapeutic IGF-1R targeting will rely on optimal combinations of IGF-1R mAbs with conventional or innovative therapies. Specifically, adoptive transfer of activated NK cells may have therapeutic benefit in Ewing sarcoma without adding toxicity. Modulatory or synergistic interactions between novel drugs and cellular therapies as a basis for potent combinations have only started to be explored. Here, we investigated the effects of IGF-1R-specific mAbs on the in vitro activation and expansion of human NK cells and their cytolytic activity against Ewing sarcoma cells. Freshly isolated PBMCs from 6 healthy donors were stimulated with irradiated K-562 in the presence or absence of two different inhibitory IGF-1R mAbs and expanded for up to 23 days. 7 of 8 NK cell cultures expanded in vitro at superior rates (3.3+/-1.2 fold) when IGF-1R mAbs were present in the cultures. These findings were reproduced in a stimulator cell free system based on magnetic cell sorting and subsequent stimulation of NK cells. Thus, IGF-1R-induced increases of NK cell expansion do not rely on interactions with bystander cells. Non-specific Fc-mediated NK cell stimulation was excluded by experiments using whole IgG as control. NK cells were found to surface-express IGF-1R and respond to coincubation with IGF-1R mAb with receptor downregulation (n=3). We conclude that direct effects of IGF-1R mAbs on the IGF-1R pathway in NK cells are likely to induce their activation and expansion. The expression of differentiation markers and activating receptors by in vitro activated and expanded NK cells was unaffected by IGF-1R antagonists. Upon coincubation with the Ewing sarcoma cell lines TC-71, TC-32 and VH-64 and with the newly established, low-passage cell culture DC-ES-6, NK cells that were activated and expanded in the presence and absence of IGF-1R antibody showed comparable, potent and reproducible degranulation responses by CD107a upregulation. Twenty-four hour preincubation of the Ewing sarcoma cell lines with IGF-1R mAb or presence of the mAbs during coculture also did not affect Ewing-sarcoma induced NK cell degranulation responses. We conclude that human NK cells respond to IGF-1R mAb inhibition with superior expansion kinetics while maintaining potent antitumor responses against Ewing sarcoma. Combining adoptive NK cell transfer with IGF-1R targeting may be an efficient means to eliminate minimal residual disease after conventional therapy and thereby rescue patients at highest risk of relapse. Citation Format: Silke Landmeier, Andrea-Caroline Krueger, Stephanie Piepke, Saskia Janneschuetz, Bianca Altvater, Sareetha Kailayangiri, Christian Spurny, Heribert Juergens, Claudia Rossig. Insulin-like growth factor-1 receptor (IGF-1R) inhibition promotes expansion of human NK cells with potent antitumor activity against Ewing sarcoma cells. [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 3974. doi:10.1158/1538-7445.AM2014-3974

Novel treatment strategies in Ewing sarcoma include molecularly targeted drugs and antibodies as well as cellular therapies. Preclinical in vivo models are needed that recapitulate the biology of multifocal disease and reflect the activity of novel therapies against systemic (micro)metastatic disease. Here, we used whole body magnetic resonance imaging techniques to monitor the engraftment and metastatic spread of human Ewing sarcoma xenografts in mice and to address the therapeutic efficacy of adoptive T cell transfer. Of 18 mice receiving intravenous injections of 2x106 VH-64 cells, all developed disseminated tumor growth detectable by whole-body MRI within 31 days. All mice had lung tumors, with a median of 19 tumors (range 1 to 60) per mouse. Sixteen mice had additional tumor manifestations, including bone and/or bone marrow (n=10), soft tissues (n=5), and kidney (n=13). Interobserver agreement was high, with an intraclass correlation of 0.929 for tumor numbers. Dissection and histological analysis confirmed the presence of CD99+ small blue round cell tumors in bones, lungs and kidneys in all examined specimens. Sequential whole body T2 MRI scans at weekly intervals following an initial scan 3 weeks after tumor inoculation revealed in vivo growth of tumors at all sites. To add further tissue information, we performed parallel diffusion weighted whole body imaging with background signal suppression (DWIBS). DWIBS effectively visualized metastatic Ewing sarcoma growth in bones, retroperitoneal organs, and soft tissues, whereas, as expected, susceptibility artifacts in air-filled spaces prevented effective detection of lung tumors. To assess the therapeutic efficacy of adoptive T cell transfer against disseminated Ewing sarcomas in this model, further cohorts of 9 mice each received transfusions of 1x107 14.G2a-28ζ gene-modified human GD2-specific T cells following tumor inoculation. Control mice received non-transduced T cells. The numbers of mice developing tumors and the numbers of tumors at extrapulmonary localizations sites were not noticeably different between treated and control mice. However, animals receiving GD2-targeted gene-modified T cell therapy had lower numbers of pulmonary tumors than controls (p Citation Format: Lennart Liebsch, Sareetha Kailayangiri, Laura Beck, Bianca Altvater, Raphael Koch, Marc Hotfilder, Janine Ring, Cornelius Faber, Volker Vieth, Claudia Rossig. Assessment of therapeutic responses of disseminated Ewing sarcoma xenografts to adoptive therapy with chimeric receptor gene-modified T cells in mice by whole body magnetic resonance imaging. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3978. doi:10.1158/1538-7445.AM2013-3978

The outcome of disseminated Ewing sarcoma remains poor despite intensive multimodal treatment regimens. The disease often responds well to chemotherapy, but systemic relapses occur in the majority of patients. Targeting of residual disease by cellular immunotherapy may sustain remission and improve outcome. Specifically, Ewing sarcoma cells have been shown to be exquisitely sensitive to targeting by activated NK cells. To further explore the value of cellular strategies, preclinical models are needed that mimic the anchorage-independent, multicellular growth of Ewing sarcoma micrometastases. Here, we generated Ewing sarcoma spheres from cell lines (VH-64, TC-32, TC-71, A4573) and from four low-passage cell cultures established from Ewing sarcoma biopsies at primary diagnosis or at relapse under serum-free growth conditions. Standard monolayer cultures were used for comparisons. Phenotypic analysis revealed considerable heterogeneity among individual Ewing sarcomas and between spheres and monolayers. While the Ewing sarcoma marker CD99 as well as CD133 were expressed at comparable densities, spheres had significantly higher expression of the neural crest marker CD57 (HNK-1) and of MHC class I than monolayers, whereas CD117 (c-kit) expression was lower. Side populations characterized by Hoechst dye exclusion and previously associated with cancer stem cell function were identified in one of two primary sphere cultures and in VH-64 spheres but were absent or reduced in monolayer cultures. However, cells resuspended from spheres did not form subcutaneous tumors in immunodeficient (NOD/scid) mice at higher efficiencies than monolayer cultures, arguing against higher tumorigenicity of sphere-cultured cells. VH64 spheres were significantly more resistant towards doxorubicine than monolayers, and resuspended cells from sphere cultures remained less susceptible to lysis than monolayer cultures, but among primary tumor cells, consistent differences in chemosensitivity were not observed between the two culture systems. In vitro activated allogeneic NK cells were uniformly capable to lyse single cells derived from both monolayer and sphere cultures from established cell lines and primary cell cultures. Moreover, NK cells efficiently eliminated intact Ewing sarcoma spheres. Thus, cultured Ewing sarcoma cells are highly heterogenous. Their phenotype, function and susceptibility to both chemo- and immunotherapy differs among individual cell lines and primary cultures and under variable in vitro growth conditions. Activated NK cells efficiently target Ewing sarcoma cells both as monolayers and as spheres. The sphere model may provide a useful tool to analyze the contribution of micrometastatic architecture and serum-free niches to immune evasion. Experiments with autologous NK cells and with NK cells engineered to express tumor antigen-specific chimeric receptors are ongoing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2503. doi:1538-7445.AM2012-2503

Primary metastatic Ewing sarcoma involving the bone and/or bone marrow has remained largely incurable. Relapses after intensive multimodal treatment regimens are attributed to a rare subpopulation of residual cells capable of reinitiating tumor growth. Cellular immunotherapy with chimeric antigen receptor (CAR) transduced T cells is a promising strategy to eradicate minimal residual disease and prevent relapse. Based on the observation that Ewing sarcoma cells express the ganglioside antigen GD2, we have explored the capacity of T cells transduced with GD2-specific CARs to functionally interact with Ewing sarcoma cells. Surface GD2 expression was detected on 10 of 11 Ewing sarcoma cell lines by flow cytometry analysis, and on 11 of 12 primary Ewing tumor samples by immunofluorescence staining. To obtain GD2-specific effector T cells, CD3/CD28 stimulated T cells from five healthy donors were retrovirally transduced with the second generation GD2-specific CAR 14.G2aCD28ζ, resulting in CAR surface expression in 83.8±2.7% (81.1-87.8%) of the cells. Ewing sarcoma cell lines expressing various levels of GD2 were used as target cell lines for 14.G2aCD28ζ-mediated cytolysis in a 51Cr release assay. Both GD2hi VH-64 and GD2intermediate Cado-ES-1 cells were efficiently lysed by gene-modified T cells (36.6±7.7% and 25.3%±8.2 lysis at a 40:1 effector-to-target cell ratio) in the absence of background cytolysis of GD2-negative tumor cells and by non-transduced T cells. Moreover, efficient and antigen-specific granzyme B secretion was found after coincubation of various Ewing sarcoma cell lines and a primary Ewing sarcoma cell culture with CAR gene-modified T cells. 14.G2aCD28ζ T cells further eradicated Ewing sarcoma cells in a serum-free spheroid model for anchorage-independent micrometastatic tumor growth. Functional activation of CAR-expressing T cells in response to GD2 positive Ewing sarcoma cells was further demonstrated by significant secretion of IFN-γ and TNFα and significant proliferative responses to various cell lines expressing low, intermediate or high levels of GD2. Thus, GD2 is a promising target antigen for the redirected antitumor activity of T cells not only in neuroblastoma and melanoma, but also in Ewing sarcoma. To address the preclinical in vivo efficacy of 14.G2aCD28ζ T cells, we have now established an in vivo model of disseminated disease by intravenous transfer of Ewing sarcoma cells to sublethally irradiated NOD/scid mice. Magnetic resonance imaging allows to visualize metastatic tumor manifestations in xenotransplanted mice. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4754. doi:10.1158/1538-7445.AM2011-4754