Non-Viral Cytokine-Inducible SH2 Containing ProteinLocus-Specific Integrated Fibroblast Activation ProteinAlpha-Targeting Chimeric Antigen Receptor T Cells AchievePotent Antitumor Efficacy in Glioblastoma featuring Full Circles Therapeutics
ABSTRACT
Chimeric antigen receptor T (CAR-T) cells have been used to treat patients with glioblastoma (GBM) in clinical trial settings bytargeting GBM-associated antigens. However, the efficacy of these CAR-T cells remains limited mainly due to the heterogeneousexpression 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 fibroblastactivation protein alpha (FAPα) as a highly attractive target for CAR-T cell therapy against GBM based on its dual expressionpattern (on tumor cells and perivascular cells) in GBM. A panel of nanobodies specific for FAPα was isolated, and FAPα-targetingCAR-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) technologywas 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 novelengineered CAR-T cells with enhanced efficacy against GBM.
