Generation of Allogeneic CAR ‐T Circumvents Functional Deficits in Patient‐Derived Autologous Product for Glioblastoma

ABSTRACT Glioblastoma (GBM) is the most common malignant brain tumor in adults, with a poor prognosis despite aggressive standard of care. Chimeric antigen receptor T‐cell (CAR‐T) therapy has shown promising results in liquid malignancies, but clinical trials in GBM targeting various tumor antigens have not shown durable clinical benefit. While this may be attributable to various tumor‐intrinsic immune evasion strategies characteristic of GBM, little work has been done to assess whether the issue is due to the quality of the CAR‐T treatment itself. Currently, CAR‐Ts for GBMs and liquid malignancies are manufactured in an autologous setting in which T‐cells are extracted from patients, engineered ex vivo, and subsequently reinfused back. However, peripheral T‐cells taken from untreated GBM patients have demonstrated qualitative and functional deficits, which may contribute to suboptimal treatment outcomes. Thus, we aimed to establish whether CAR‐Ts generated from GBM patients would show reduced efficacy in comparison to healthy donors using our previously validated CD133 CAR‐T. In this work, we show pre‐treatment exhaustion and reduced survival advantage in autologous, patient‐derived CD133‐targeting CAR‐T cell products using an orthotopic xenograft model of human GBM. To overcome the functional and logistical considerations of autologous therapy, we additionally aimed to generate an “off‐the‐shelf” allogeneic CD133 CAR‐T. Using CRISPR gene editing technology, we generated TCR‐knockout CAR‐T cells with comparable pre‐clinical efficacy to our autologous models. Ultimately, this work highlights the need to reassess autologous CAR‐T therapy for GBM and consider allogeneic approaches as biologically informed therapeutic alternatives.