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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.

Rhabdomyosarcomas are the most frequent malignant soft tissue tumors of childhood; however, because current multimodality treatments fail to improve the poor survival rate of children with metastatic rhabdomyosarcoma, new treatments are required. We previously identified the γ-subunit of the fetal acetylcholine receptor (fAChR) as a specific cell surface target in rhabdomyosarcoma. Here, we engineered human T lymphocytes to express chimeric receptors composed of the antigen-binding domain of a human anti-fAChR antibody joined to the signaling domain of the human T-cell receptor ζ-chain. The interaction of fAChRζ-transduced T cells with fAChR-positive rhabdomyosarcoma cell lines, but not with fAChR-negative control cells, induced T-cell activation characterized by strong secretion of IFN-γ and delayed lysis of tumor cells. Importantly, we found that in six of six rhabdomyosarcoma patients, chemotherapy increased fAChR expression on residual tumor cells in vivo. Our observations suggest that these fully human chimeric fAChRζ-transduced T cells, which should be well tolerated by the patient, have potential use in vivo both as a primary treatment for rhabdomyosarcoma and as a complementary approach to eradicate residual tumor cells after chemotherapy. (Cancer Res 2006; 66(1): 24-28)

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.

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.

The efficacy of current cancer vaccines is limited by the functional heterogeneity and poor availability and expansion of professional antigen-presenting cells (APCs). Besides their potent innate effector properties, gammadelta T cells have been suggested to be involved in the initiation and maintenance of adaptive immune responses. Here, we investigated the capacity of human gammadelta T cells to induce expansion of virus-specific T cells to Epstein Barr virus (EBV) antigens. Aminobisphosphonate-stimulated human peripheral blood-derived gammadelta T cells (Vgamma2+Vdelta2+) acquired a dual phenotype characteristic for both APCs and effector memory T cells. Coincubation of activated gammadelta T cells pulsed with human leukocyte antigen-restricted epitopes of either the highly stimulatory EBV lytic cycle antigen Bam H1 Z fragment leftward open reading frame or the tumor-associated latent EBV antigen latent membrane protein 2a (LMP2a) with autologous peripheral blood lymphocytes induced selective expansion of peptide-specific, fully functional CD3CD8 cytolytic effector memory T cells. Furthermore, gammadelta T APCs efficiently processed and presented endogenous antigen, as demonstrated by the capacity of LMP2a gene-transduced gammadelta T cells to induce expansion of T cells with broad specificity for various LMP2a peptides. The capacity of autologous gammadelta T cells to induce LMP2a-specific autologous cytotoxic T lymphocytes was confirmed in 2 patients with Hodgkin lymphoma. In summary, bisphosphonate-activated human gammadelta T cells stimulate expansion of cytotoxic effector T cells specific for both subdominant and dominant viral epitopes and thus show promise as a novel source of efficient APCs for immunotherapy of viral and malignant disease.

T cells with grafted specificities for surface antigens provide an avenue for rapidly producing immune effector cells with tumor specificity. However, the function of chimeric receptor (chRec) gene-modified T cells is limited by lack of T-cell expansion and persistence. We propose to use varicella zoster virus (VZV)–reactive T cells as host for the chRec because these cells can be expanded both in vitro and in vivo by stimulation of their native receptor during endogenous reexposure to the virus or by administration of VZV vaccine. We obtained human T cells reactive with VZV from the peripheral blood of seropositive donors by stimulation with VZV lysate and evaluated their characteristics after genetic modification with two tumor-specific model chRecs. Cultures dominated by cytolytic CD4+ T cells (VZV-CTL) could be expanded and maintained in vitro. Gene-modified VZV-CTL recognized and lysed tumor targets in a MHC-independent manner while maintaining functional, MHC-restricted interaction with VZV antigen through their native receptor. Thus, chRec-transduced VZV-CTL may provide a source of potent tumor-reactive cells for adoptive immunotherapy of cancer. The availability of a safe and effective VZV vaccine provides the option of repeated in vivo stimulation to maintain high T-cell numbers until the tumor is eliminated. [Cancer Res 2007;67(17):8335–43]

The Na+/proline transporter PutP of Escherichia coli is a member of a large family of Na+/substrate symporters. Previous work on PutP suggests an involvement of the region ranging from Asp-55 to Gly-58 in binding of Na+ and/or proline (Pirch, T., Quick, M., Nietschke, M., Langkamp, M., Jung, H. (2002) J. Biol. Chem. 277, 8790-8796). In this study, a complete Cys scanning mutagenesis of transmembrane domain II (TM II) of PutP was performed to further elucidate the role of the TM in the transport process. Strong defects of PutP function were observed upon substitution of Ala-48, Ala-53, Trp-59, and Gly-63 by Cys in addition to the previously characterized residues Asp-55, Ser-57, and Gly-58. However, except for Asp-55 none of these residues proved essential for function. The activity of eight mutants was sensitive to N-ethylmaleimide inhibition with the sensitive positions clustering predominantly on a hydrophilic face in the cytoplasmic half of TM II. The same face was also highly accessible to the bulky sulfhydryl reagent fluorescein 5-maleimide in randomly oriented membrane vesicles, suggesting an unrestricted accessibility of the corresponding amino acid positions via an aqueous pathway. Na+ stimulated the reactivity of Cys toward fluorescein 5-maleimide at two positions while proline inhibited reaction of the sulfhydryl group at nine positions. Taken together, the results demonstrate that TM II of PutP is of particular functional importance. It is proposed that hydrophilic residues in the cytoplasmic half of TM II participate in the formation of an aqueous cavity in the membrane that allows Na+ and/or proline binding to residues located in the middle of the TM (e.g. Asp-55 and Ser-57). In addition, the data indicate that TM II participates in Na+- and proline-induced conformational alterations.

Cytotoxic T cells transduced with chimeric anti-CD19 receptors prevent engraftment of primary lymphoblastic leukemia in vivo

Immunosuppressive CD4+CD25(hi)FoxP3+ T cells (T(reg) cells) have been found at increased densities within the tumor microenvironment in many malignancies and interfere with protective antitumor immune responses. Osseous Ewing sarcomas (ESs) are thought to derive from a bone marrow (BM) mesenchymal cell of origin, and microscopic marrow involvement defines a subpopulation of patients at a high risk of relapse. We hypothesized that BM-resident T cells may contribute to a permissive milieu for immune escape of ESs. Using 6-color-flow cytometry, we investigated the pattern of immune cell subset distribution including NK cells, gammadelta T cells, central and effector memory CD8+ and CD4+ T cells as well as T cells with regulatory phenotype (T(reg) cells) in BM obtained at diagnosis from 45 primary or relapsed ES patients treated within standardized protocols. Although patients at relapse had an inverted CD4:CD8 T-cell ratio, neither CD8+ effector/memory T-cell subsets nor T(reg) cells significantly differed from patients at diagnosis. No significant associations of innate and effector/memory T-cell subpopulations with known risk factors were found, including age, gender, tumor site, primary metastases and histological tumor response. By contrast, T(reg) cells were found at significantly higher frequencies in patients with primary metastatic disease compared with localized ESs (5.0 vs. 3.3%, p = 0.01). Thus, increased BM T(reg) cells in patients with metastasized ES may reflect an immune escape mechanism that contributes to the development of metastatic disease. Immunotherapeutic strategies will have to adequately consider the regulatory milieu within areas of Ewing tumor-immune interactions.

Regulatory NK cell receptors can contribute to antigen-specific adaptive immune responses by modulating T cell receptor (TCR)-induced T cell activation. We investigated the potential of the NK cell receptor 2B4 (CD244) to enhance tumor antigen-induced activation of human T cells. 2B4 is a member of the CD2 receptor subfamily with both activating and inhibitory functions in NK cells. In T cells, its expression is positively associated with the acquisition of a cytolytic effector memory phenotype. Recombinant chimeric receptors that link extracellular single-chain Fv fragments specific for the tumor-associated surface antigens CD19 and G(D2) to the signaling domains of human 2B4 and/or TCRzeta were expressed in non-specifically activated peripheral blood T cells by retroviral gene transfer. While 2B4 signaling alone failed to induce T cell effector functions or proliferation, it significantly augmented the antigen-specific activation responses induced by TCRzeta. 2B4 costimulation did not affect the predominant effector memory phenotype of expanding T cells, nor did it increase the proportion of T cells with regulatory phenotype (CD4+CD25(hi)FoxP3+). These data support a costimulatory role for 2B4 in human T cell subpopulations. As an amplifier of TCR-mediated signals, 2B4 may provide a powerful new tool for immunotherapy of cancer, promoting sustained activation and proliferation of gene-modified antitumor T cells.

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

SummaryExpression of tumour antigen-specific chimaeric receptors in T lymphocytes can redirect their effector functions towards tumour cells. Integration of the signalling domains of the co-stimulatory molecule CD28 into chRec enhances antigen-specific proliferation of polyclonal human T cell populations. While CD28 plays an essential role in the priming of naive CD4+ T cells, its contribution to effector memory T cell responses is controversial. We compared the function of the chRec with and without the CD28 co-stimulatory domain, expressing it in peripheral blood T cells or Epstein–Barr virus (EBV)-specific T cell lines. The chimaeric T cell receptors contain an extracellular single-chain antibody domain, to give specificity against the tumour ganglioside antigen GD2. The transduced cytotoxic T lymphocytes (CTL) maintained their specificity for autologous EBV targets and their capacity to proliferate after stimulation with EBV-infected B cells. Intracellular cytokine staining demonstrated efficient and comparable antigen-specific interferon (IFN)-γ secretion by CTL following engagement of both the native and the chimaeric receptor, independent of chimaeric CD28 signalling. Furthermore, tumour targets were lysed in an antigen-specific manner by both chRec. However, while antigen engagement by CD28ζ chRec efficiently induced expansion of polyclonal peripheral blood lymphocytes in an antigen-dependent manner, CD28 signalling did not induce proliferation of EBV–CTL in response to antigen-expressing tumour cells. Thus, the co-stimulatory requirement for the efficient activation response of antigen-specific memory cells cannot be mimicked simply by combining CD28 and ζ signalling. The full potential of this highly cytolytic T cell population for adoptive immunotherapy of cancer requires further exploration of their co-stimulatory requirements.

BACKGROUND No effective therapeutic modalities exist for the treatment of relapsed high risk acute lymphoblastic leukemia (ALL). Adoptive cellular immunotherapy by transfusion of polyclonal donor lymphocytes is not always effective and is limited by cellular cross-reactivity with normal tissues, leading to development of clinical graft-versus-host disease (GVHD). METHOD To develop an immunotherapeutic strategy for targeted elimination of residual leukemic blasts, human T cells were gene-modified to express CD19-specific chimeric receptors. RESULTS Gene-modified T cells specifically lyse CD19-expressing lymphatic blast cells, however, they show a limited proliferative response to stimulation with CD19. Integration of the signal transduction domain of the costimulatory molecule CD28 enhances the proliferative properties of the gene-modified T cells. CONCLUSIONS Adoptive transfer of gene-modified virus-specific T cells may provide a useful strategy for prevention and early treatment of ALL relapses following allogeneic stem cell transplantation.

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

High-risk Ewing sarcoma is still fatal in many cases. Cellular immunotherapy is a promising approach for preventing relapse by eliminating residual disease after conventional treatment. A critical prerequisite is the availability of an adequate tumor target antigen. STEAP-1 is a surface protein aberrantly expressed in various cancers. Potential obstacles to the use of STEAP-1 as a tumor target are the limited immunogenicity of STEAP-1 epitopes, clonal deletion of STEAP-1-reactive T cells, and poor presentation of STEAP-1 peptides by MHC class I on tumor cells. Here we investigated whether functional and Ewing-tumor reactive STEAP-1-specific cytotoxic T cells (CTLs) can be expanded from the repertoire of normal donors by peptide stimulation. STEAP-1 was confirmed to be expressed in 10 of 10 Ewing sarcoma cell lines by PCR. To expand STEAP-1-specific CTLs, CD8+ T cells from HLA-A2-positive healthy donors were stimulated with autologous dendritic cells pulsed with a pool of 4 STEAP-1 peptides (5 µM each), in the presence of rhIL-7, rhIL-12 and rhIL-15. Weekly restimulations were performed with peptide-pulsed K562 cells gene-modified to express human HLA-A2, CD80, CD40L and OX40L (K562aAPCs). CTLs with a predominant CD8+ effector memory CTL phenotype (CD45RA-, CCR7-) were successfully generated from 6 healthy donors. Their epitope specificity was confirmed by ELISPOT analysis after 2 to 6 rounds of restimulation. Pooled STEAP-1 peptides directly added to the CTLs induced specific secretion of IFN-γ (70 to 487 spot forming cells (SFC)/105 cells, mean 297.5±138.7 SFC/105 cells), in the absence of relevant background responses to a control peptide (0 to 50 and mean of 16.2±19.9 SFCs/105 cells). Restimulation of CTLs with individual STEAP-1 peptides demonstrated donor-dependent reactivity with one to four of the peptides, while neither one emerged as immunodominant. The CTLs specifically lysed STEAP-1 peptide pulsed target cells (52.9±4.3% at an effector-to-target ratio of 20:1), but not cells pulsed with control peptides (0.0±3.8%). In contrast, STEAP-1 specific CTLs failed to functionally interact with the HLA-A2+/STEAP-1+ Ewing sarcoma cell lines as measured by cytolysis and cytokine secretion, even after upregulation of MHC class I molecules by pretreatment with IFN-γ. Thus, while the induction of STEAP-1 peptide-specific CTLs is feasible, these CTLs do not efficiently recognize endogenously expressed antigen on Ewing sarcoma cells. Higher-avidity CTLs are likely needed for exploiting STEAP-1 as a target for adoptive immunotherapy in this disease. To define the critical requirements for recognition and lysis of STEAP-1 expressing target cells in an autologous setting, we are currently exploring activated autologous γδ T-APCs expressing full-length STEAP-1 protein by retroviral gene transfer as targets and stimulator cells for STEAP-1 specific CTLs. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1927.

Besides its role in hemato- and lymphopoiesis, bone marrow (BM) has emerged as a secondary lymphoid organ with important roles in both T cell priming and memory responses. Due to these properties, non-malignant T cells persisting within the BM of patients with acute leukemias may be involved in the immune response to leukemia and the control of minimal residual disease. Here, we investigated the phenotypic signature of residual T cells present at diagnosis in 25 pediatric patients (age 2–16 years) with B cell precursor ALL. Patients with high risk disease including Philadelphia chromosome-positive or MLL-rearranged leukemias were excluded from this analysis. Mononuclear cells were isolated from freshly aspirated BM by density gradient centrifugation and analyzed by six-color-flow cytometry using monoclonal antibodies directed towards various T-cell associated surface and intracellular markers, including CD3/CD56/TCRαβ/TCRγδ/CD86/HLA-DR (Panel 1); CD3/CD4/CD8/CCR7/CD45RA/ CD45RO (Panel 2); CD4/FoxP3/CD25/CD45RA/CD45RO/CCR7 (Panel 3). For each sample, ≥15,000 CD3+ cells (Panel 1, 2) or ≥8,000 CD4+ cells (Panel 3) were analyzed with FACS Canto and Diva Software. The Student’s t test was used to determine statistical significances between individual subgroups, and correlations were performed using the Pearson test. Consistent with published data on BM T cell subsets in healthy donors, the CD4+/CD8+ T cell ratio was 1.32±0.41. The predominant subset among CD8+ T cells (55.2±17.6%) had a naïve T cell phenotype (CD45RA+CCR7+), while 20.7±11.5% were effector memory T cells (TEM; CD45RA-CCR7-), and 7.1±6.2% were central memory T cells (TCM; CD45RA-CCR7+). No differences were found between TEL/AML1 positive or negative leukemias, or between patients stratified into standard (SR) vs. medium risk (MR) groups according to the criteria of the ALL-BFM 2000 study group. T cells bearing γδ T cell receptors have been attributed important roles in the primary immune defense against microbes and in immune control of cancer. We found that 6.9±3.0% (1.8 to 11.8%) of BM T cells were γδTCR+ (Vγ9Vδ2). A statistically significant (p

Genetic modification of polyclonal T cells with tumor antigen-specific chimeric receptors (chRec) specifically redirects their effector functions towards tumor cells. However, the therapeutic value of chRec-gene-modified T cells is limited, since T cell activation by chRec fails to mediate proliferative responses, probably due to the lack of costimulatory T cell signaling in response to tumor cells. The costimulatory signaling domain of CD28 has been shown to enhance chimeric-receptor mediated proliferation in polyclonal T cell populations but not in CD8+ effector memory CTL, which represent the primary mediators of effective antitumor immunity. Indeed, alternative costimulatory molecules are now recognised as having complementary roles in the activation of antigen-experienced effector T cells. The SLAM-related receptor 2B4 (CD244), which is expressed on NK cells and on CD8+ cytotoxic T cells, is known to positively regulate T-cell-mediated cytotoxicity. We hypothesized that inclusion of the 2B4 endodomain into the chRec would enhance tumor-specific activation and proliferation of in vitro expanded effector T cells. We focused our studies on a GD2-specific scFv, which is currently being used in clinical trials for the treatment of neuroblastoma, and generated chRecs containing either the 2B4 signaling domain alone (14.G2a-2B4) or combined with TCR (14.G2a-2B4ζ). These chRecs were expressed in activated polyclonal human peripheral blood T cells using retroviral gene transfer. As controls, T cells were transduced with 14.G2aζ. High chRec surface expression was obtained for all three constructs, with 51±12% for 14.G2a-ζ, 70±15% for 14.G2a-2B4, and 53±7% for 14.G2a-2B4ζ. Immunphenotypes were dominated by a CD3+CD8+ population in all cell cultures. 51Cr-release assays showed efficient and comparable lysis of GD2+ tumor targets by T cells transduced with 14.G2a-ζ (44±10%) and 14.G2a-2B4ζ (44±3%) at an effector-to-target ratio of 40:1, whereas 2B4 alone failed to mediate specific tumor cell lysis. Intracellular cytokine secretion by chRec+ T cells was induced in response to GD2+ tumor targets by 14.G2a-ζ (mean 18.3%, range 12.1–29.7% IFN-γ+ CD3+ T cells) and 14.G2a-2B4ζ (mean 7.7%, range 6.0–11.3%). In contrast, 14.G2a-2B4 transduced T cells failed to induce IFN-γ secretion (mean 0.2%, range 0.06–0.47%). Weekly stimulations with tumor cells resulted in substantially superior expansion of T cells transduced with 14.G2a-2B4ζ (8–45fold) compared to T cells transduced with either 14.G2a-ζ (3.5–18fold) or 14.G2a-2B4 (2–4fold) over a 5 week period. It is known that T cell costimulation by CD28 receptor family members is mediated by downstream signaling involving the NFκB pathway. However in our studies, luciferase reporter gene assays failed to show a significant difference in tumor antigen-specific NFκB recruitment between T cells transduced with 14.G2aζ (240,000±5,000 RLU) and 14.G2a-2B4ζ (180,000±7,000 RLU). Although the nature of the signaling pathway mediating 2B4-induced T cell stimulation is still to be defined, our data support a costimulatory role for the activating NK cell receptor 2B4 in peripheral blood T cells. Therefore, 2B4-containing chRec may be a powerful new tool for adoptive immunotherapy of cancer.

Efficient antigen presentation is an important prerequisite for the induction of T cell mediated immunity against viral and tumor antigens. The functional heterogeneity, limited availability and poor expansion of dendritic cells (DC) has motivated a search for alternative sources of antigen-presenting cells (APC). Bisphosphonate-activated human γδ T cells were shown to function as APC by inducing primary T cell responses to allogeneic and microbial antigens. Here, we extended these observations by investigating the capacity of activated γδ T cells to present Epstein Barr virus lytic cycle and latency antigens in a peptide-specific manner and induce expansion of fully functional, virus-specific cytotoxic αβ T cells. Peripheral blood-derived γδ T cells were expanded from three individual healthy donors by stimulation with the aminobisphosphonate zoledronic acid (1 μg/ml) in the presence of rhIL-2 (100 U/ml) and rhIL-15 (10 ng/ml). Under these conditions, γδ T cells (Vγ9+Vδ2+) acquired a phenotype characteristic for APC, including upregulation of HLA-DR as well as the costimulatory ligands CD80, CD83, CD86, CD40, and 4-1BBL (CDw137L). Whereas expression of most markers decreased after peak levels were reached on day 5, CD86 and HLA-DR remained upregulated on >80% of the cells for prolonged culture periods of >14 days. We next assessed the APC function of activated γδ T cells by testing their ability to stimulate expansion of antigen-specific cytotoxic T cells in vitro. Autologous peripheral blood lymphocytes (PBMC) were incubated with activated γδ T cells pulsed with the HLA-B8-restricted epitope of the EBV lytic cycle antigen BZLF1 (RAKFKQLL). We demonstrated selective expansion of RAK-pentamer-specific CD3+ CD8+ T cells to a mean of 15% (range 6–24%) on day 10. BZLF-1 represents a highly immunogenic virus antigen, whereas the EBV latency-associated antigen LMP-2a is less T cell-stimulatory and, due to its expression on Hodgkin′s lymphoma cells, is an important target for tumor-specific immune therapy. Stimulation of PBMC with γδ T cells pulsed with the HLA-A2-restricted LMP2a epitope FLYALALLL also resulted in a substantial increase of pentamer-reactive T cells to a mean of 3.5% (1.4–5.5%), which was comparable to the increase obtained when peptide-loaded autologous DC were used (mean of 3.5%; range 1.2–5.7%). γδ T cell stimulation in the absence of peptide failed to induce specific T cell expansion (mean of 0.1%; 0.03–0.15%). CD8+ T cells expanded against EBV-peptide-pulsed activated γδ T cells functionally interacted with peptide-loaded autologous DC or lymphoblastoid cell lines (LCL), as demonstrated by efficient and specific MHC class I-restricted cytolysis of 41–65% at an effector-target ratio of 40:1. Peptide-specific cytotoxic T cell functionality was comparable to that obtained with T cells expanded in the presence of pulsed autologous DC from the same donors. In summary, bisphosphonate-activated γδ T cells are potent antigen-presenting cells for reproducible expansion of disease antigen-specific CTL. Thus, they represent a novel source of highly efficient professional APC for antigen-specific immunotherapy of viral or malignant disease.

Adoptive transfer of gene-engineered T cells expressing tumor antigen-specific chimeric receptors (chRec) is a promising tool in cancer immunotherapy. A major limitation is the failure of chRec to induce proliferative T cell responses, resulting in a rapid loss of function. To provide a strategy for reexpansion of tumor-reactive T cells in vivo, we generated dual-specific T cells that respond to varicella zoster virus while also possessing chRec-mediated tumor reactivity. We expanded VZV-specific cytotoxic T cell lines (VZV-CTL) from four seropositive donors by culturing peripheral blood-derived T cells with lysates extracted from VZV-infected fibroblasts. Repeated stimulation with VZV lysates resulted in efficient and continued expansion for 10–12 weeks. >1x109 T cells were routinely obtained from a starting number of 1x106 peripheral blood T cells. The T cells displayed a mainly CD3+CD4+ (90±5%) phenotype. ELISPOT assays showed specific, MHC class II-restricted IFN-γ release in response to CD40-activated B cells expressing the viral glycoproteins gE and IE62. VZV-CTL belong to a non-regulatory effector T cell subset, shown by their failure to exert antiproliferative effects against cocultured autologous T cells and lack of Foxp3 expression. Retroviral transduction with chRec recognizing the tumor ganglioside antigen GD2 (14.G2a-ζ) and the B cell lineage antigen CD19 (CD19-ζ) resulted in receptor surface expression on 29–74% and 39–45% of cells, respectively. Gene-modified VZV-CTL efficiently recognized antigen-expressing tumor targets in an MHC-independent manner, as demonstrated by antigen-specific secretion of IFN-γ in response to coincubation with GD2-expressing tumor targets. Furthermore, chRec-transduced VZV-CTL performed potent and antigen-specific tumor cytolysis. Antibody blocking experiments revealed that tumor cells were lysed in a granulysin-dependent manner. ChRec-transduced CD3+CD4+ cytolytic VZV-CTL may provide a source of highly potent tumor-reactive cells for adoptive immunotherapy cancer. Endogenous viral reactivations or administration of booster doses of varicella vaccine may lead to survival of these tumor-reactive T cells for prolonged periods of time in vivo.

Abstract 3025 Poster Board II-1001 Due to its restriction to the B-cell lineage and high surface expression in B-cell malignancies, CD19 is an attractive target antigen for immunological strategies in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). While preclinical in vivo studies of CD19-specific cellular immunotherapy have generally used xenografts from human CD19+ leukemia cell lines, primary leukemia cells are likely to more closely mimic the disease in humans and allow to differentiate between standard and high risk situations. Therefore, we investigated the in vivo sensitivity of human leukemic bone marrow to adoptive immunotherapy with gene-modified CD19-specific T cells. Among 15 primary leukemias obtained from the bone marrow of pediatric patients at diagnosis, 10 were successfully engrafted in NOD/scid mice by intrafemoral injection within 6 to 20 weeks. For therapeutic experiments, we focused on one standard risk leukemia, characterized by a rapid and sustained response to multiagent chemotherapy, and on a leukemia bearing the high-risk feature of an MLL rearrangement, which was refractory to standard treatment. Titration experiments demonstrated reliable engraftment of 1×104 leukemic cells per mouse. For CD19-directed T-cell therapy, cytotoxic T cells (CTLs) with native specificity for Epstein-Barr virus antigens were expanded from 4 healthy donors and transduced to express either a codon-optimized CD19-specific chimeric antigen receptor (CAR) containing the intracellular signaling domain of the TCRz chain (CD19-z), or a control CAR directed against the neuroectodermal antigen GD2 (14.G2a-z). Costimulatory domains now commonly used to ensure sustained T-cell activation via CARs were not included, since previous studies have shown that CAR activity in virus-specific CTLs does not benefit from additional signaling elements. CTLs had a uniform CD8+ effector memory T-cell phenotype (CD45RO+, CCR7-), and CAR surface expression was 73±21%, range 32-93% (CD19-z, n=9) and 18±13%, range 6-35% (14.G2a-z, n=5). In vitro cytotoxicity experiments confirmed specific lysis of the CD19+ leukemia cell lines REH (51Cr release 59.7±7.2% at an effector target ratio of 20:1) and SupB15 (66.7±8.6) as well as primary CD19+ leukemic cells from 5 pediatric patients (47.2±13.2%), in the absence of background lysis by 14.G2a-z-transduced control CTLs. 1×104 leukemic cells per mouse from primary engrafted mice were transferred into further cohorts of NOD/scid mice by secondary intrafemoral transplantation, followed by adoptive transfer of 4 doses of 5×106 CTLs via tail vein injection on days 1, 4, 8, and 11. IL-2 (500 IU/mouse) was administered twice-weekly, and sequential murine bone marrow aspirates were analyzed for human leukemia engraftment by flow cytometry using human CD45 and CD19-specific antibodies starting 3 weeks after transplantation. CD19z CTLs prevented engraftment of the standard risk leukemia in 3 of 4 mice, while 3 of 4 control mice developed the leukemia (p = 0.158, Log Rank/Mantel-Cox Test). Moreover, while the MLL-rearranged human leukemia became detectable in the bone marrow of 4 of 5 control mice, followed by overt and fatal leukemia, 5 of 8 mice receiving transfusions of CD19-z transduced CTLs remained disease-free (p = 0.067), and 6 of 8 remained alive, one of them with detectable leukemia cells (p = 0.054) (see Figure). Thus, adoptive transfer of CD19-redirected CTLs efficiently delayed or prevented engraftment of both standard and high risk ALLs in mice and therefore provides a promising treatment option for patients with BCP-ALL refractory to standard treatment. Disclosures No relevant conflicts of interest to declare.