Glioblastoma (GBM), which is the most common malignant brain tumor, is resistant to standard treatments. Immunotherapy might be a promising alternative for the treatment of this cancer. Chimeric antigen receptor (CAR) is an artificially modified fusion protein that can be engineered to direct the specificity and function of T cells against tumor antigens. However, the antitumor effects of EGFRvIII-targeting CAR-T (EvCAR-T) cells in GBM are limited. The inhibitory effect is induced by the interaction between programmed cell death protein 1 (PD-1) on activated EvCAR-T cells and its ligands on GBM cells. In the present study, PD-1-disrupted EvCAR-T cells were established using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9). The sgRNA/Cas9 expression vectors designed precisely disrupted the target region of PD-1 and inhibited the expression of PD-1 in EvCAR-T cells. The PD-1-disrupted EvCAR-T cells had an in vitro growth inhibitory effect on EGFRvIII-expressing GBM cells without altering the T-cell phenotype and the expression of other checkpoint receptors. In the future, the in vivo antitumor effect of this vector should be evaluated in order to determine if it could be applied clinically for improving the efficacy of EvCAR-T cell-based adoptive immunotherapy for GBM.

Glioblastoma (GBM) is the most common and aggressive primary malignant brain tumor in adults. Natural Killer (NK) cells are potent cytotoxic effector cells against tumor cells inducing GBM cells; therefore, NK cell based- immunotherapy might be a promising target in GBM. T cell immunoglobulin mucin family member 3 (TIM3), a receptor expressed on NK cells, has been suggested as a marker of dysfunctional NK cells. We established TIM3 knockout in NK cells, using the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9). Electroporating of TIM3 exon 2- or exon 5-targeting guide RNA- Cas9 protein complexes (RNPs) inhibited TIM3 expression on NK cells with varying efficacy. T7 endonuclease I mutation detection assays showed that both RNPs disrupted the intended genome sites. The expression of other checkpoint receptors, i.e., programmed cell death 1 (PD1), Lymphocyte-activation gene 3 (LAG3), T cell immunoreceptor with Ig and ITIM domains (TIGIT), and TACTILE (CD96) were unchanged on the TIM3 knockout NK cells. Real time cell growth assays revealed that TIM3 knockout enhanced NK cell–mediated growth inhibition of GBM cells. These results demonstrated that TIM3 knockout enhanced human NK cell mediated cytotoxicity on GBM cells. Future, CRISPR-Cas9 mediated TIM3 knockout in NK cells may prove to be a promising immunotherapeutic alternative in patient with GBM.

Glioblastoma (GBM) is the most aggressive and malignant primary brain tumor in adults. Despite multimodality treatment involving surgical resection, radiation therapy, chemotherapy, and tumor-treating fields, the median overall survival (OS) after diagnosis is approximately 2 years and the 5-year OS is poor. Considering the poor prognosis, novel treatment strategies are needed, such as immunotherapies, which include chimeric antigen receptor T-cell therapy, immune checkpoint inhibitors, vaccine therapy, and oncolytic virus therapy. However, these therapies have not achieved satisfactory outcomes. One reason for this is that these therapies are mainly based on activating T cells and controlling GBM progression. Natural killer (NK) cell-based immunotherapy involves the new feature of recognizing GBM via differing mechanisms from that of T cell-based immunotherapy. In this review, we focused on NK cell-based immunotherapy as a novel GBM treatment strategy.

Glioblastoma is the leading malignant glioma with a poor prognosis. This study aimed to investigate the antitumor effects of natural killer cells in combination with temozolomide as the standard chemotherapeutic agent for glioblastoma. Using a simple, feeder-less, and chemically defined culture method, we expanded human peripheral blood mononuclear cells and assessed the receptor expression, natural killer cell activity, and regulatory T cell frequency in expanded cells. Next, using the standard human glioblastoma cell lines (temozolomide-sensitive U87MG, temozolomide-resistant T98G, and LN-18), we assessed the ligand expressions of receptors on natural killer cells. Furthermore, the antitumor effects of the combination of the expanded natural killer cells and temozolomide were assessed using growth inhibition assays, apoptosis detection assays, and senescence-associated β-galactosidase activity assays in the glioblastoma cell lines. Novel culture systems were sufficient to attain highly purified (>98%), expanded (>440-fold) CD3⁻/CD56⁺ peripheral blood-derived natural killer cells. We designated the expanded population as genuine induced natural killer cells. Genuine induced natural killer cells exhibited a high natural killer activity and low regulatory T cell frequency compared with lymphokine-activated killer cells. Growth inhibition assays revealed that genuine induced natural killer cells inhibited the glioblastoma cell line growth but enhanced temozolomide-induced inhibition effects in U87MG. Apoptosis detection assays revealed that genuine induced natural killer cells induced apoptosis in the glioblastoma cell lines. Furthermore, senescence-associated β-galactosidase activity assays revealed that temozolomide induced senescence in U87MG. Genuine induced natural killer cells induce apoptosis in temozolomide-sensitive and temozolomide-resistant glioblastoma cells and enhances temozolomide-induced antitumor effects in different mechanisms. Hence, the combination of genuine induced natural killer cells and temozolomide may prove to be a promising immunochemotherapeutic approach in patients with glioblastoma if the antitumor effects in vivo can be demonstrated.

Cell-based immunotherapy is categorized as a regenerative therapy under the Regenerative Medicine Safety Act in Japan. Natural killer (NK) cell-based immunotherapy is considered a promising strategy for treating cancer, including glioblastoma (GBM). We previously reported an expansion method for highly purified human peripheral blood-derived NK cells using a cytokine cocktail. Here, we aimed to establish a more efficient NK cell expansion method as compared to our previously reported method. T cell-depleted human peripheral blood mononuclear cells (PBMCs) were isolated from three healthy volunteers. The depleted PBMCs were cultured in the presence of recombinant human interleukin (rhIL)-18 and high-dose rhIL-2 in anti-NKp46 and/or anti-CD16 antibody immobilization settings. After 14 days of expansion, the purity and expansion ratio of CD3-CD56+ NK cells were determined. The cytotoxicity-mediated growth inhibition of T98G cells (an NK activity-sensitive GBM cell line) was evaluated using a non-labeling, impedance-based real-time cell analyzer. Anti-NKp46 stimulation increased the NK cell purity and expansion ratio as compared to the non-antibody-stimulated population. Anti-CD16 stimulation weakly enhanced the NK cell expansion ratio of the non-antibody-stimulated population and enhanced the NK cell purity and expansion ratio of anti-NKp46-stimulated populations. All NK cell-containing populations tested distinctly inhibited T98G cell growth. These effects tended to be enhanced in an NK cell purity-dependent manner. In some cases, anti-CD16 stimulation decreased growth inhibition of T98G cell compared to other conditions despite the comparable NK cell purity. We established a robust large-scale feeder-free expansion system for highly purified human NK cells using a defined cytokine cocktail and anti-NK cell activating receptor antibodies. The expansion system could be feasible for autologous or allogeneic NK cell-based immunotherapy of GBM. Moreover, it is easily controlled under Japanese law on regenerative medicine.