Non‐Viral Cytokine‐Inducible SH2 Containing Protein Locus‐Specific Integrated Fibroblast Activation Protein Alpha‐Targeting Chimeric Antigen Receptor T Cells Achieve Potent Antitumor Efficacy in Glioblastoma

ABSTRACT Chimeric antigen receptor T (CAR‐T) cells have been used to treat patients with glioblastoma (GBM) in clinical trial settings by targeting GBM‐associated antigens. However, the efficacy of these CAR‐T cells remains limited mainly due to the heterogeneous expression of tumor antigen and their anergy in the tumor microenvironment (TME). Cytokine‐inducible SH2‐containing protein (CIS, encoded by the gene CISH ) is a potent intracellular checkpoint inducing T‐cell anergy. Here, we identified fibroblast activation protein alpha (FAPα) as a highly attractive target for CAR‐T cell therapy against GBM based on its dual expression pattern (on tumor cells and perivascular cells) in GBM. A panel of nanobodies specific for FAPα was isolated, and FAPα‐targeting CAR‐T cells were developed using the isolated nanobody to verify their specific cytotoxicity to GBM cells. Furthermore, a non‐viral circular single‐stranded DNA (cssDNA)‐based CRISPR/Cas9‐targeted genome‐editing (cssDNA/CRISPR/Cas9) technology was used to integrate CAR cassettes at the CISH locus to generate CISH ‐knockout ( CISH ‐KO) CAR‐T cells. The resulting CISH ‐KO‐CAR‐T cells exhibited robust proliferation and potent anti‐GBM activity in vitro and in vivo. Thus, our results provide novel engineered CAR‐T cells with enhanced efficacy against GBM.