Sareetha Kailayangiri

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.

Activated human γδ T cells induce peptide-specific CD8+ T-cell responses to tumor-associated self-antigens

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.

Targeting Ewing sarcoma with activated and GD2-specific chimeric antigen receptor-engineered human NK cells induces upregulation of immune-inhibitory HLA-G

ABSTRACT 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.

Anchorage-independent growth of Ewing sarcoma cells under serum-free conditions is not associated with stem-cell like phenotype and function

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.

Insulin-like growth factor-1 receptor (IGF-1R) inhibition promotes expansion of human NK cells which maintain their potent antitumor activity against Ewing sarcoma cells

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.

Common Ewing sarcoma-associated antigens fail to induce natural T cell responses in both patients and healthy individuals

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.

Programmed cell death ligand 1 (PD‐L1) expression is not a predominant feature in Ewing sarcomas

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.

Abstract 458: Immune-inhibitory HLA-G is expressed in the tumor microenvironment of Ewing Sarcomas

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA 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

Abstract 3974: Insulin-like growth factor-1 receptor (IGF-1R) inhibition promotes expansion of human NK cells with potent antitumor activity against Ewing sarcoma cells

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

Abstract 3978: 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.

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 2x10 6 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 1x10 7 14.G2a-28ζ gene-modified human G D2 -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 G D2 -targeted gene-modified T cell therapy had lower numbers of pulmonary tumors than controls (p D2 -redirected T cells had a growth delay of their lung tumors, with both smaller volumes (p=0.023) and lower numbers of tumors (p=0.024) at 4 weeks after tumor inoculation. Thus, G D2 -retargeted T cells cannot prevent disseminated tumor growth in this aggressive systemic disease model, but are active to reduce pulmonary Ewing sarcoma manifestations. Optimized strategies now aim to enhance the efficacy of chimeric T cell receptor therapies. 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