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.

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.

Glioblastoma (GBM) is the most common and aggressive primary brain tumor, with a dismal prognosis. Natural killer (NK) cells are large granular lymphocytes with natural cytotoxicity against tumor cells, and they should be established for the novel treatment of patients with GBM. We previously reported highly activated, and ex vivo-expanded NK cells derived from human peripheral blood, designated genuine induced NK cells (GiNK), which were induced by specific culture conditions and which exerted a cytotoxic effect on GBM cells via apoptosis. Here, we comprehensively summarize the molecular characteristics, especially focusing on the expression of stem cell markers, extracellular matrix markers, chemokines, chemokine receptors, and NK receptor ligands of spheroids derived from GBM cell lines as compared with that of two-dimensional (2D) adherent GBM cells via microarray. The spheroid had upregulated gene expression of stem cell markers, extracellular matrix markers, chemokines, chemokine receptors, and NK cell inhibitory receptor ligands compared with the 2D adherent GBM cells. Preclinical evaluation of the NK cells was performed via an ex vivo 3D spheroid model derived from GBM cell lines. In the model, the NK cells accumulated and infiltrated around the spheroids and induced GBM cell death. Flow cytometry-based apoptosis detection clearly showed that the NK cells induced GBM cell death via apoptosis. Our findings could provide pivotal information for NK cell-based immunotherapy for patients with GBM.

Glioblastoma (GBM) is the leading malignant intracranial tumor and is associated with a poor prognosis. Highly purified, activated natural killer (NK) cells, designated as genuine induced NK cells (GiNKs), represent a promising immunotherapy for GBM. We evaluated the anti-tumor effect of GiNKs in association with the programmed death 1(PD-1)/PD-ligand 1 (PD-L1) immune checkpoint pathway. We determined the level of PD-1 expression, a receptor known to down-regulate the immune response against malignancy, on GiNKs. PD-L1 expression on glioma cell lines (GBM-like cell line U87MG, and GBM cell line T98G) was also determined. To evaluate the anti-tumor activity of GiNKs in vivo, we used a xenograft model of subcutaneously implanted U87MG cells in immunocompromised NOG mice. The GiNKs expressed very low levels of PD-1. Although PD-L1 was expressed on U87MG and T98G cells, the expression levels were highly variable. Our xenograft model revealed that the retro-orbital administration of GiNKs and interleukin-2 (IL-2) prolonged the survival of NOG mice bearing subcutaneous U87MG-derived tumors. PD-1 blocking antibodies did not have an additive effect with GiNKs for prolonging survival. GiNKs may represent a promising cell-based immunotherapy for patients with GBM and are minimally affected by the PD-1/PD-L1 immune evasion axis in GBM.

Glioblastoma (GBM) is a malignant brain tumor with a poor prognosis. The standard postoperative chemotherapy is temozolomide (TMZ), which does not greatly improve survival. The DNA repair gene O-6-met...