Key Points GD2-specific CAR renders NKT cells cytotoxic against NB cells and results in potent in vivo antitumor activity without graft-versus-host disease. The 4-1BB-containing CAR constructs induce T helper 1–like polarization in NKT cells.

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Vα24-invariant natural killer T (NKT) cells have shown potent anti-tumor properties in murine tumor models and have been linked to favorable outcomes in patients with cancer. However, low numbers of these cells in humans have hindered their clinical applications. Here we report interim results from all three patients enrolled on dose level 1 in a phase 1 dose-escalation trial of autologous NKT cells engineered to co-express a GD2-specific chimeric antigen receptor (CAR) with interleukin-15 in children with relapsed or resistant neuroblastoma (NCT03294954). Primary and secondary objectives were to assess safety and anti-tumor responses, respectively, with immune response evaluation as an additional objective. We ex vivo expanded highly pure NKT cells (mean ± s.d., 94.7 ± 3.8%) and treated patients with 3 × 10 6 CAR-NKT cells per square meter of body surface area after lymphodepleting conditioning with cyclophosphamide/fludarabine (Cy/Flu). Cy/Flu conditioning was the probable cause for grade 3-4 hematologic adverse events, as they occurred before CAR-NKT cell infusion, and no dose-limiting toxicities were observed. CAR-NKT cells expanded in vivo, localized to tumors and, in one patient, induced an objective response with regression of bone metastatic lesions. These initial results suggest that CAR-NKT cells can be expanded to clinical scale and safely applied to treat patients with cancer.

Purpose: Va24-invariant natural killer T cells (NKT) are attractive carriers for chimeric antigen receptors (CAR) due to their inherent antitumor properties and preferential localization to tumor sites. However, limited persistence of CAR-NKTs in tumor-bearing mice is associated with tumor recurrence. Here, we evaluated whether coexpression of the NKT homeostatic cytokine IL15 with a CAR enhances the in vivo persistence and therapeutic efficacy of CAR-NKTs.Experimental Design: Human primary NKTs were ex vivo expanded and transduced with CAR constructs containing an optimized GD2-specific single-chain variable fragment and either the CD28 or 4-1BB costimulatory endodomain, each with or without IL15 (GD2.CAR or GD2.CAR.15). Constructs that mediated robust CAR-NKT cell expansion were selected for further functional evaluation in vitro and in xenogeneic mouse models of neuroblastoma.Results: Coexpression of IL15 with either costimulatory domain increased CAR-NKT absolute numbers. However, constructs containing 4-1BB induced excessive activationinduced cell death and reduced numeric expansion of NKTs compared with respective CD28-based constructs. Further evaluation of CD28-based GD2.CAR and GD2. CAR.15 showed that coexpression of IL15 led to reduced expression levels of exhaustion markers in NKTs and increased multiround in vitro tumor cell killing. Following transfer into mice bearing neuroblastoma xenografts, GD2.CAR.15 NKTs demonstrated enhanced in vivo persistence, increased localization to tumor sites, and improved tumor control compared with GD2.CAR NKTs. Importantly, GD2.CAR.15 NKTs did not produce significant toxicity as determined by histopathologic analysis.Conclusions: Our results informed selection of the CD28based GD2.CAR.15 construct for clinical testing and led to initiation of a first-in-human CAR-NKT cell clinical trial (NCT03294954).

Vα24-invariant NKT cells inhibit tumor growth by targeting tumor-associated macrophages (TAMs).

Hepatocellular carcinoma (HCC) is the fourth most common cause of cancer-related death in the world, and curative systemic therapies are lacking. Chimeric antigen receptor (CAR)-expressing T cells induce robust antitumor responses in patients with hematologic malignancies but have limited efficacy in patients with solid tumors, including HCC. IL15 and IL21 promote T-cell expansion, survival, and function and can improve the antitumor properties of T cells. We explored whether transgenic expression of IL15 and/or IL21 enhanced glypican-3-CAR (GPC3-CAR) T cells' antitumor properties against HCC. We previously optimized the costimulation in GPC3-CARs and selected a second-generation GPC3-CAR incorporating a 4-1BB costimulatory endodomain (GBBz) for development. Here, we generated constructs encoding IL15, IL21, or both with GBBz (15.GBBz, 21.GBBz, and 21.15.GBBz, respec-tively) and examined the ability of transduced T cells to kill, produce effector cytokines, and expand in an antigen-dependent manner. We performed gene-expression and phenotypic analyses of GPC3-CAR T cells and CRISPR-Cas9 knockout of the TCF7 gene. Finally, we measured GPC3-CAR T-cell antitumor activity in murine xenograft models of GPC3 þ tumors. The increased proliferation of 21.15.GBBz T cells was at least in part dependent on the upregulation and maintenance of TCF-1 (encoded by TCF7) and associated with a higher percentage of stem cell memory and central memory populations after manufacturing. T cells expressing 21.15. GBBz had superior in vitro and in vivo expansion and persistence, and the most robust antitumor activity in vivo. These results provided preclinical evidence to support the clinical evaluation of 21.15.GPC3-CAR T cells in patients with HCC.

Neuroblastoma is a neural crest derived embryonal malignancy which accounts for 13% of all pediatric cancer mortality, primarily due to tumor recurrence. Therapy-resistant cancer stem cells are implicated in tumor relapse, but definitive phenotypic evidence of the existence of these cells has been lacking. In this study, we define a highly tumorigenic subpopulation in neuroblastoma with stem cell characteristics, based on the expression of CD114, which encodes the receptor for granulocyte colony-stimulating factor (G-CSF). CD114+ cells isolated from a primary tumor and the NGP cell line by flow cytometry were highly tumorigenic and capable of both self-renewal and differentiation to progeny cells. CD114+ cells closely resembled embryonic and induced pluripotent stem cells with respect to their profiles of cell cycle, microRNA and gene expression. In addition, they reflect a primitive undifferentiated neuroectodermal/neural crest phenotype revealing a developmental hierarchy within neuroblastoma tumors. We detected this de-differentiated neural crest subpopulation in all established neuroblastoma cell lines, xenograft tumors, and primary tumor specimens analyzed. Ligand activation of CD114 by the addition of exogenous G-CSF to CD114+ cells confirmed intact STAT3 upregulation, characteristic of G-CSF receptor signaling. Together our data describe a novel distinct subpopulation within neuroblastoma with enhanced tumorigenicity and a stem-cell like phenotype, further elucidating the complex heterogeneity of solid tumors such as neuroblastoma. We propose this subpopulation may represent an additional target for novel therapeutic approaches to this aggressive pediatric malignancy.

Mucosal delivery of vaccines against sexually transmitted pathogens is important to elicit strong immune responses at biologically relevant sites. However, inclusion of appropriate adjuvants is essential to overcome the inherent mucosal tolerance. We present evidence in support of the effectiveness of co-administering alpha-Galactosylceramide (α-GalCer) as an adjuvant with a CTL-inducing HIV envelope peptide, via either oral or intranasal route, to prime antigen-specific immune responses in multiple systemic and mucosal compartments. Contrary to the known potential of repeated parenteral dosing with a-GalCer to induce NKT cell anergy that could compromise adoptive immunity development, we have observed that two and three doses delivered by the intranasal or oral route were more efficient in priming broader antigen-specific immune responses. These results demonstrate the effectiveness of α-GalCer as adjuvant for repeated intranasal or oral administration of vaccines for protection against mucosally transmitted pathogens.