Effect of CRISPR/Cas9-mediated PD-1-disrupted primary human third-generation CAR-T cells targeting EGFRvIII on human glioblastoma cell growth

Abstract

Abstract Glioblastoma (GBM) is the most common and malignant brain tumor. As GBM is resistant to standard treatment, immunotherapy might be a promising alternative. 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. The antitumor effects of EGFRvIII-targeting CAR-T (EvCAR-T) cells are limited in GBM, on account of the interaction between programmed cell death protein 1 (PD-1) on activated EvCAR-T cells and its ligands on target tumor cells. Antibodies against PD-1 have shown limitations, so permanent deletion of the PD-1 gene in CAR-T cells might be a promising solution. Therefore, in the present study, PD-1-disrupted EvCAR-T cells were established for the first time with the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene-editing tool. We determined the disruption efficiency of the human PD-1 gene in EvCAR-T cells and evaluated the antitumor effect of PD-1-disrupted EvCAR-T cells against human GBM cells. The results showed that our designed-sgRNA/Cas9 expression vectors precisely disrupted the target region of PD-1 and inhibited the expression of PD-1 in EvCAR-T cells. Further, the PD-1-disrupted EvCAR-T cells also had a growth inhibitory effect on EGFRvIII-expressing GBM cells. Thus, this study provides a simple, easy, and clinical applicable strategy for inducing PD-1-disrupted EvCAR-T cells, which could potentially be useful for improving the efficacy of EvCAR-T cell-based adoptive immunotherapy for GBM, and even other cancers.