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Non-viral CISH locus-specific integrated FAPα CAR-T cells achieve potent anti-tumor efficacy in glioblastoma

ABSTRACT: 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 remain limited mainly due to heterogeneous expression of tumor antigen and their anergy in 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 were then isolated using a yeast surface display library. FAPα-targeting CAR-T cells were developed using the isolated nanobody and verified for 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 CISH locus to generate CISH-knockout (CISH-KO) CAR-T cells. The resulted CISH-KO CAR-T cells exhibited robust proliferation and potent anti-GBM activity in vitro and in vivo compared with conventional lentivirus-transduced CAR-T cells. Our proof-of-concept study demonstrates that CISH-KO FAPα-targeting CAR-T cells engineered by non-viral locus-specific integration represent a promising therapeutic approach for GBM.

ORGANISM(S): Homo sapiens

PROVIDER: GSE314496 | GEO | 2025/12/25

REPOSITORIES: GEO

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Project description:A significant challenge for chimeric antigen receptor (CAR) T cell therapy against glioblastoma (GBM) is its immunosuppressive tumor microenvironment (TME), which is densely populated and supported by protumoral glioma-associated microglia and macrophages (GAMs). Targeting CD47, a don't-eat-me signal overexpressed by tumor cells, disrupts the CD47-SIRPalpha axis and induces GAM phagocytic function. However, antibody-mediated CD47 blockade monotherapy is associated with toxicity and low bioavailability in solid tumors. To overcome these limitations, we combined local CAR T cell therapy with paracrine GAM modulation to effectively eliminate GBM. To this end, we engineered a new CAR T cell against epidermal growth factor receptor variant III (EGFRvIII) that constitutively secretes a signal regulatory protein gamma (SIRPgamma)-related protein (SGRP) with high affinity to CD47. Anti-EGFRvIII-SGRP CAR T cells eliminated EGFRvIII+ GBM in a dose-dependent manner in vitro and eradicated orthotopically xenografted EGFRvIII-mosaic GBM by locoregional application in vivo. This resulted in significant tumor-free long-term survival, followed by partial tumor control upon tumor re-challenge. Combining anti-CD47 antibodies with anti-EGFRvIII CAR T cells failed to achieve a similar therapeutic effect, underscoring the importance of sustained paracrine GAM modulation. Multidimensional brain immunofluorescence microscopy and in-depth spectral flow cytometry on GBM-xenografted brains showed that anti-EGFRvIII-SGRP CAR T cells accelerated GBM clearance, increased CD68+ cell trafficking to tumor scar sites and promoted GAM-mediated tumor cell uptake. In a peripheral lymphoma mouse xenograft model, anti-CD19-SGRP CAR T cells had superior efficacy to conventional anti-CD19 CAR T cells. Validation on human GBM explants revealed that anti-EGFRvIII-SGRP CAR T cells had a similar tumor-killing capacity to anti-EGFRvIII CAR monotherapy but showed a slight improvement in the maintenance of tumor-infiltrated CD14+ cells. Thus, local anti-EGFRvIII-SGRP CAR T cell therapy combines the potent antitumor effect of engineered T cells with the modulation of the surrounding innate immune TME. This results in the additive elimination of bystander EGFRvIII- tumor cells in a manner that overcomes the main mechanisms of CAR T cell therapy resistance, including tumor innate immune suppression and antigen escape.

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Non-viral CISH locus-specific integrated FAPα CAR-T cells achieve potent anti-tumor efficacy in glioblastoma

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