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 G D2 -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.

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.

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.

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.