In this paper, we present the role of autologous and allogeneic monocytes from healthy individuals and those of the cancer patients, with a number of distinct cancers, in activating the function of natural killer (NK) cells, in particular, in induction of IFN-γ secretion by the NK cells and the functional capability of secreted IFN-γ in driving differentiation of the tumor cells. In addition, we compared the roles of CD16 signaling as well as sonicated probiotic bacteria AJ2 (sAJ2)-mediated induction and function of IFN-γ-mediated differentiation in tumor cells. We found that monocytes from cancer patients had lower capability to induce functional IFN-γ secretion by the autologous CD16 mAb-treated NK cells in comparison to those from healthy individuals. In addition, when patient monocytes were cultured with NK cells from healthy individuals, they had lower capability to induce functional IFN-γ secretion by the NK cells when compared to those from autologous monocyte/NK cultures from healthy individuals. Activation by sAJ2 or addition of monocytes from healthy individuals to patient NK cells increased the secretion of functional IFN-γ by the NK cells and elevated its functional capability to differentiate tumors. Monocytes from cancer patients were found to express lower CD16 receptors, providing a potential mechanism for their lack of ability to trigger secretion of functional IFN-γ. In addition to in vitro studies, we also conducted in vivo studies in which cancer patients were given oral supplementation of AJ2 and the function of NK cells were studied. Oral ingestion of AJ2 improved the secretion of IFN-γ by patient derived NK cells and resulted in the better functioning of NK cells in cancer patients. Thus, our studies indicate that for successful NK cell immunotherapy, not only the defect in NK cells but also those in monocytes should be corrected. In this regard, AJ2 probiotic bacteria may serve to provide a potential adjunct treatment strategy.

Natural killer (NK) cells are the key immune effectors with the ability to mediate selection and differentiation of a number of different cancer stem cells/undifferentiated tumors via lysis, and secreted or membrane-bound interferon (IFN)-γ and tumor necrosis factor (TNF)-α, respectively, leading to curtailment of tumor growth and metastasis. In this review, we present an overview of our recent findings on the biology and significance of NK cells in selection and differentiation of stem-like tumors using in vitro and in vivo studies conducted in humanized-BLT mice and in cancer patients. In addition, we present current advances in NK cell expansion and therapeutic delivery, and discuss the utility of allogeneic supercharged NK cells in the treatment of cancer patients. Moreover, we discuss the potential loss of NK cell numbers and function at the neoplastic and pre-neoplastic stages of tumorigenesis in induction and progression of pancreatic cancer. Therefore, because of their indispensable role in targeting cancer stem-like/undifferentiated tumors, NK cells should be placed high in the armamentarium of tumor immunotherapy. A combination of allogeneic supercharged NK cells with other immunotherapeutic strategies such as oncolytic viruses, antibody-dependent cellular cytotoxicity (ADCC)-inducing antibodies, checkpoint inhibitors, chimeric antigen receptor (CAR) T cells, CAR NK cells, and chemotherapeutic and radiotherapeutic strategies can be used for the ultimate goal of tumor eradication.

Unlimited generation of chimeric antigen receptor (CAR) T cells from human-induced pluripotent stem cells (iPSCs) is an attractive approach for "off-the-shelf" CAR T cell immunotherapy. Approaches to efficiently differentiate iPSCs into canonical αβ T cell lineages, while maintaining CAR expression and functionality, however, have been challenging. We report that iPSCs reprogramed from CD62L⁺ naive and memory T cells followed by CD19-CAR engineering and 3D-organoid system differentiation confers products with conventional CD8αβ-positive CAR T cell characteristics. Expanded iPSC CD19-CAR T cells showed comparable antigen-specific activation, degranulation, cytotoxicity, and cytokine secretion compared with conventional CD19-CAR T cells and maintained homogeneous expression of the TCR derived from the initial clone. iPSC CD19-CAR T cells also mediated potent antitumor activity in vivo, prolonging survival of mice with CD19⁺ human tumor xenografts. Our study establishes feasible methodologies to generate highly functional CAR T cells from iPSCs to support the development of "off-the-shelf" manufacturing strategies.

Chimeric antigen receptor T cell (CAR-T) therapy is an emerging strategy to improve treatment outcomes for recurrent high-grade glioma, a cancer that responds poorly to current therapies. Here we report a completed phase I trial evaluating IL-13Rα2-targeted CAR-T cells in 65 patients with recurrent high-grade glioma, the majority being recurrent glioblastoma (rGBM). Primary objectives were safety and feasibility, maximum tolerated dose/maximum feasible dose and a recommended phase 2 dose plan. Secondary objectives included overall survival, disease response, cytokine dynamics and tumor immune contexture biomarkers. This trial evolved to evaluate three routes of locoregional T cell administration (intratumoral (ICT), intraventricular (ICV) and dual ICT/ICV) and two manufacturing platforms, culminating in arm 5, which utilized dual ICT/ICV delivery and an optimized manufacturing process. Locoregional CAR-T cell administration was feasible and well tolerated, and as there were no dose-limiting toxicities across all arms, a maximum tolerated dose was not determined. Probable treatment-related grade 3+ toxicities were one grade 3 encephalopathy and one grade 3 ataxia. A clinical maximum feasible dose of 200 × 10⁶ CAR-T cells per infusion cycle was achieved for arm 5; however, other arms either did not test or achieve this dose due to manufacturing feasibility. A recommended phase 2 dose will be refined in future studies based on data from this trial. Stable disease or better was achieved in 50% (29/58) of patients, with two partial responses, one complete response and a second complete response after additional CAR-T cycles off protocol. For rGBM, median overall survival for all patients was 7.7 months and for arm 5 was 10.2 months. Central nervous system increases in inflammatory cytokines, including IFNγ, CXCL9 and CXCL10, were associated with CAR-T cell administration and bioactivity. Pretreatment intratumoral CD3 T cell levels were positively associated with survival. These findings demonstrate that locoregional IL-13Rα2-targeted CAR-T therapy is safe with promising clinical activity in a subset of patients. ClinicalTrials.gov Identifier: NCT02208362 .