Integrative analysis of neuroblastoma by single-cell RNA sequencing identifies the NECTIN2-TIGIT axis as a target for immunotherapy.

Pediatric patients with high-risk neuroblastoma have poor survival rates and urgently need more effective treatment options with less side effects. Since novel and improved immunotherapies may fill this need, we dissect the immunoregulatory interactions in neuroblastoma by single-cell RNA-sequencing of 24 tumors (10 pre- and 14 post-chemotherapy, including 5 pairs) to identify strategies for optimizing immunotherapy efficacy. Neuroblastomas are infiltrated by natural killer (NK), T and B cells, and immunosuppressive myeloid populations. NK cells show reduced cytotoxicity and T cells have a dysfunctional profile. Interaction analysis reveals a vast immunoregulatory network and identifies NECTIN2-TIGIT as a crucial immune checkpoint. Combined blockade of TIGIT and PD-L1 significantly reduces neuroblastoma growth, with complete responses (CR) in vivo. Moreover, addition of TIGIT+PD-L1 blockade to standard relapse treatment in a chemotherapy-resistant Th-ALKF1174L/MYCN 129/SvJ syngeneic model induces CR. In conclusion, our integrative analysis provides promising targets and a rationale for immunotherapeutic combination strategies.

Three-Dimensional Modeling of Solid Tumors and Their Microenvironment to Evaluate T Cell Therapy Efficacy In Vitro.

Immunotherapy development for solid tumors remains challenging, partially due to a lack of reproducible, cost-effective in vitro three-dimensional (3D) models to mimic the heterogeneous and complex tumor microenvironment. Here, we investigate the cellular anti-tumor reactivity of αß T cells engineered to express a defined γδ TCR (TEG A3). For that purpose, we developed a 3D cytotoxicity assay targeting cell line-derived spheroids or patient-derived tumor organoids formed in serum-free media. Tumor cell lysis by TEG A3 was monitored using the Incucyte S3 live-cell imaging system with the apoptosis marker caspase 3/7 green and endpoint readouts of IFN-γ secretion in the supernatant. The 3D cytotoxicity assay model system was able to adequately demonstrate TEG A3 reactivity toward targets expressing an isoform of CD277 (CD277J). To obtain a more complex heterogeneous tumor microenvironment, patient-derived organoids were mixed with unmatched patient-derived fibroblasts or matched cancer-associated fibroblasts. In all assays, we demonstrated the tumor target specificity of TEG A3, lysing tumor cells within 48 h. Our study demonstrates the utility of complex 3D cytotoxicity assay model systems incorporating the tumor microenvironment in the functional evaluation of T cell-based adoptive immunotherapy, providing a useful platform for early-stage preclinical development of immunotherapies.

Epigenetic modulation of neuroblastoma enhances T cell and NK cell immunogenicity by inducing a tumor-cell lineage switch.

BACKGROUND: Immunotherapy in high-risk neuroblastoma (HR-NBL) does not live up to its full potential due to inadequate (adaptive) immune engagement caused by the extensive immunomodulatory capacity of HR-NBL. We aimed to tackle one of the most notable immunomodulatory processes in neuroblastoma (NBL), absence of major histocompatibility complex class I (MHC-I) surface expression, a process greatly limiting cytotoxic T cell engagement. We and others have previously shown that MHC-I expression can be induced by cytokine-driven immune modulation. Here, we aimed to identify tolerable pharmacological repurposing strategies to upregulate MHC-I expression and therewith enhance T cell immunogenicity in NBL. METHODS: Drug repurposing libraries were screened to identify compounds enhancing MHC-I surface expression in NBL cells using high-throughput flow cytometry analyses optimized for adherent cells. The effect of positive hits was confirmed in a panel of NBL cell lines and patient-derived organoids. Compound-treated NBL cell lines and organoids were cocultured with preferentially expressed antigen of melanoma (PRAME)-reactive tumor-specific T cells and healthy-donor natural killer (NK) cells to determine the in vitro effect on T cell and NK cell cytotoxicity. Additional immunomodulatory effects of histone deacetylase inhibitors (HDACi) were identified by transcriptome and translatome analysis of treated organoids. RESULTS: Drug library screening revealed MHC-I upregulation by inhibitor of apoptosis inhibitor (IAPi)- and HDACi drug classes. The effect of IAPi was limited due to repression of nuclear factor kappa B (NFκB) pathway activity in NBL, while the MHC-I-modulating effect of HDACi was widely translatable to a panel of NBL cell lines and patient-derived organoids. Pretreatment of NBL cells with the HDACi entinostat enhanced the cytotoxic capacity of tumor-specific T cells against NBL in vitro, which coincided with increased expression of additional players regulating T cell cytotoxicity (eg, TAP1/2 and immunoproteasome subunits). Moreover, MICA and MICB, important in NK cell cytotoxicity, were also increased by entinostat exposure. Intriguingly, this increase in immunogenicity was accompanied by a shift toward a more mesenchymal NBL cell lineage. CONCLUSIONS: This study indicates the potential of combining (immuno)therapy with HDACi to enhance both T cell-driven and NKcell-driven immune responses in patients with HR-NBL.

αß-T Cells Engineered to Express γδ-T Cell Receptors Can Kill Neuroblastoma Organoids Independent of MHC-I Expression.

Currently ~50% of patients with a diagnosis of high-risk neuroblastoma will not survive due to relapsing or refractory disease. Recent innovations in immunotherapy for solid tumors are highly promising, but the low MHC-I expression of neuroblastoma represents a major challenge for T cell-mediated immunotherapy. Here, we propose a novel T cell-based immunotherapy approach for neuroblastoma, based on the use of TEG002, αß-T cells engineered to express a defined γδ-T cell receptor, which can recognize and kill target cells independent of MHC-I. In a co-culture killing assay, we showed that 3 out of 6 neuroblastoma organoids could activate TEG002 as measured by IFNγ production. Transcriptional profiling showed this effect correlates with an increased activity of processes involved in interferon signaling and extracellular matrix organization. Analysis of the dynamics of organoid killing by TEG002 over time confirmed that organoids which induced TEG002 activation were efficiently killed independent of their MHC-I expression. Of note, efficacy of TEG002 treatment was superior to donor-matched untransduced αß-T cells or endogenous γδ-T cells. Our data suggest that TEG002 may be a promising novel treatment option for a subset of neuroblastoma patients.