# 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

## TL;DR

Researchers developed a new type of CAR-T cell targeting FAPα in glioblastoma, which showed improved tumor-fighting ability by integrating the CAR at a specific gene locus.

## Contribution

A novel non-viral CRISPR-based method to generate CISH-knockout CAR-T cells targeting FAPα for enhanced glioblastoma treatment.

## Key findings

- CISH-KO FAPα-CAR-T cells showed robust proliferation and potent anti-GBM activity in vitro and in vivo.
- FAPα-targeting CAR-T cells using a high-affinity nanobody demonstrated superior antitumor efficacy compared to conventional CAR-T cells.

## 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.

FAPα is a dual target on glioblastoma cells and perivascular cells. Using a high‐affinity anti‐FAPα nanobody, we generated CISH‐KO FAPα‐CAR‐T cells via non‐viral CRISPR–Cas9 integration. These cells demonstrated enhanced proliferation and superior antitumor efficacy in patient‐derived organoids and orthotopic mouse models compared to conventional CAR‐T cells. Image created with BioRender.com with permission.