Activated and expanded natural killer (NK) cells have substantial cytotoxicity against many tumor cells, but their 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 G in activated NK cells increased their responses to G+ allogeneic EwS cells 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 G-specific CAR gene-modified NK cells both by intratumoral and intraperitoneal delivery failed to eliminate G-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, 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. 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.

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-Gpos 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-Gneg 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-Gneg. 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: 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.

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.

To enhance the potency of chimeric antigen receptor (CAR) engineered T cells in solid cancers, we designed a novel cell-based combination strategy with an additional therapeutic mode of action. CAR T cells are used as micropharmacies to produce a targeted pro-coagulatory fusion protein, truncated tissue factor (tTF)-NGR, which exerts pro-coagulatory activity and hypoxia upon relocalization to the vascular endothelial cells that invade tumor tissues. Delivery by CAR T cells aimed to induce locoregional tumor vascular infarction for combined immune-mediated and hypoxic tumor cell death. Human T cells that were one-vector gene-modified to express a GD2-specific CAR along with CAR-inducible tTF-NGR exerted potent GD2-specific effector functions while secreting tTF-NGR that activates the extrinsic coagulation pathway in a strictly GD2-dependent manner. In murine models, the CAR T cells infiltrated GD2-positive tumor xenografts, secreted tTF-NGR into the tumor microenvironment and showed a trend towards superior therapeutic activity compared with control cells producing functionally inactive tTF-NGR. In vitro evidence supports a mechanism of hypoxia-mediated enhancement of T cell cytolytic activity. We conclude that combined CAR T cell targeting with an additional mechanism of antitumor action in a one-vector engineering strategy is a promising approach to be further developed for targeted treatment of solid cancers.

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

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<0.01 for CD8+ T cells). In conclusion, we found that the stimulation conditions have a strong impact on the T cell phenotype and that retroviral CAR gene transduction can also affect T cell differentiation. The optimal T cell culture conditions for a product with sustained persistence in vivo will ultimately emerge from clinical trials. Table 1:Proportions of T cell subpopulations on day 13 or 14 (medians and ranges)xyTN: CD45RO-/CD197+TCM: CD45RO+/CD197+TEM: CD45RO+/CD197-3/28NT37.9% CD4+ (31.8-41.3) 53.0% CD8+ (20.9-72.9)20.2% CD4+ (20.2-27.0) 6.1% CD8+ (3.7-6.9)35.2% CD4+ (32.8-45.4) 35.5% CD8+ (17.0-66.3)CAR d224.4% CD4+ (15.1-28.8) 32.9% CD8+ (23.9-48.9)33.6% CD4+ (29.9-49.3) 27.7% CD8+ (17.7-38.8)37.3% CD4+ (22.4-47.2) 27.1% CD8+ (25.4-32.7)DBNT33.1% CD4+ (15.0-45.6) 53.8% CD8+ (46.8-53.8)32.6% CD4+ (29.9-45.2) 19.6% CD8+ (17.8-26.3)23.3% CD4+ (16.6-51.7) 12.1% CD8+ (10.6-15.2)CAR d211.5% CD4+ (10.9-27.7) 37.1% CD8+ (24.7-41.860.7% CD4+ (53.0-69.7) 48.0% CD8+ (34.4-65.2)16.5% CD4+ (16.1-26.6) 8.7% CD8+ (5.7-10.3)DBNT33.2% CD4+ (26.5-55.2) 51.6% CD8+ (34.2-62.5)23.0% CD4+ (21.9-40.2) 14.2% CD8+ (11.2-16.9)19.1% CD4+ (17.6-48.6) 15.2% CD8+ (13.0-19.6)CAR d317.5% CD4+ (13.4-34.5) 36.6% CD8+ (28.5-46.5)45.7% CD4+ (36.6-57.5) 38.0% CD8+ (22.2-55.1)22.0% CD4+ (21.4-32.6) 10.8% CD8+ (8.5-19.6) 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.

CAR T cell therapy of solid tumors is challenged by the heterogeneity of target expression and by mechanical and immune-modulatory barriers in the tumor microenvironment (TME). To combine CAR-retargeted T cell effector functions with a second therapeutic mode of action, we designed an innovative cell-based combination strategy. CAR-engineered antitumor effector T cells are used as micropharmacies to produce and deliver a pro-coagulatory fusion protein, tTF-NGR, in the TME to induce locoregional tumor vascular infarction for combined T-cell mediated and hypoxic tumor cell death. tTF-NGR is a CD13-targeted tissue factor variant with coagulation activity upon relocalization into the phospholipid membranes of the CD13-expressing vascular endothelial cells that invade tumor tissues. Consequent thrombosis in tumor blood vessels induces tumor infarction, growth retardation and regression in preclinical in vitro and in vivo studies and selective reduction of tumor blood flow in a clinical phase I study. Human T cells were co-transduced by retroviral one-vector gene transfer to express genes encoding for a GD2-specific CAR and for tTF-NGR, the latter in an antigen-dependent CAR-mediated manner. The engineered T cells exerted potent GD2 antigen-specific effector functions, including secretion of IFN-γ and TNF-α, upregulation of CD107 and tumor cell lysis, comparable to control CAR T cells producing mutant tTF-NGR lacking pro-coagulatory function. They secreted recombinant tTF-NGR in a strictly antigen-dependent manner upon coincubation with the anti-idiotype antibody ganglidiomab, which selectively engages the extracellular scFv of the CAR, or with GD2-positive tumor cells, shown by ELISA. tTF-NGR produced by human T cells effectively activates the extrinsic coagulation cascade, thus it retains its pro-coagulatory activity. In a murine Ewing sarcoma xenograft model which expresses the CAR target GD2on tumor cells along with CD13 on tumor vascular endothelial cells, GD2-specific CAR T cells with inducible tTF-NGR had noticeably superior therapeutic activity compared with control cells excreting mutant tTF-NGR. Mechanistic evidence hints at hypoxia-induced higher CAR T cell cytolytic activity. We conclude that combined CAR-mediated T cell targeting of cancer cells with CD13-targeted vascular infarction of the TME in a one-vector engineering strategy is a promising approach to overcome limitations of both strategies for effective targeting and eradication of solid cancers. Citation Format: Bianca Altvater, Sareetha Kailayangiri, Christian Spurny, Maike Flügge, Jutta Meltzer, Lea Greune, Christian Schwöppe, Caroline Brand, Christoph Schliemann, Wolfgang Hartmann, Hinrich Abken, Axel Schambach, Nicole Farwick, Wolfgang E. Berdel, Claudia Rossig. CAR T cells as micropharmacies to induce locoregional tumor vascular infarction by antigen-specific delivery of tissue factor to the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3182.