Glioblastoma (GBM) is the most aggressive and lethal primary brain tumor in adults, characterized by resistance to standard therapies, including surgical resection, radiation, chemotherapy, and targeted agents. While chimeric antigen receptor (CAR)-engineered T (CAR-T) cell therapy has emerged as a promising immunotherapeutic approach for GBM, its application remains limited by tumor antigen escape, an immunosuppressive tumor microenvironment (TME), treatment-associated toxicities such as cytokine release syndrome (CRS), and the logistical complexities of autologous cell manufacturing. In this study, we leveraged hematopoietic stem and progenitor cell (HSPC) gene engineering combined with a feeder-free, ex vivo differentiation protocol to generate allogeneic EGFRvIII-specific CAR-engineered invariant natural killer T (ECAR-NKT) cells through a clinically guided, scalable platform. These cells exhibit potent, multifaceted antitumor activity against GBM, including direct tumor cell killing via CAR and NK receptors and selective targeting of CD1dimmunosuppressive cells within the TME via their invariant T cell receptors. In both subcutaneous and orthotopic GBM humanized models,ECAR-NKT cells demonstrated robust efficacy, minimal systemic leakage from the brain, and a reduced risk of CRS. Collectively, our findings supportECAR-NKT cells as a next-generation, off-the-shelf immunotherapy with enhanced efficacy and safety for the treatment of GBM. Allo + Allo Allo
