246 Disruption of the cytokine signaling checkpoint CIS enhances serial-killing and anti-tumor activity of CAR-engineered γδ T cells

Background

γδ T cells have emerged as a promising allogeneic cell-based immunotherapy platform for the treatment of cancer. Armed with a unique combination of innate and adaptive immunity to target malignant cells, the infiltration and presence of γδ T cells in various solid tumors is significantly correlated with survival benefit. Additional modifications to engineer γδ T cells to express CARs have shown enhanced tumoricidal activity and encouraging clinical efficacy. Opportunities to understand the mechanisms that drive these potent anti-tumor responses in γδ T cells will be important to further augment their effector functions. Cytokine-inducible SH2-containing protein (CIS) is a key negative regulator of IL-15 and TCR signaling.^{1 2} CIS disruption in αβ T cells and NK cells promotes increased persistence and anti-tumor activity, but the role of CIS in γδ T cells is not well understood. Here, we report, for the first time, a strategy that couples targeting of the CIS loci within allogeneic CAR engineered γδ T cells for enhanced anti-tumor potential.

Methods

Using CRISPR gene-editing methods, the CISH gene was knocked out in γδ T cells expanded from PBMCs of healthy donors. CISH KO γδ T cells were transduced to express a CAR targeting the B7-H6 protein. CISH gene knockout (KO) was confirmed by indel sequencing, western blot, and flow cytometry. In vitro phenotype, proliferation, and antitumor activity of CISH KO CAR Vδ1 T cells were measured using flow cytometry and cell-based cytotoxicity assays against different tumor cell lines. Supernatants from co-cultures with tumor cells were analyzed for cytokine levels using the Luminex^® Multiplex Assay, and the Nanostring nCounter^® analysis system was used to evaluate gene expression.

Results

CRISPR-Cas gene editing of Vδ1 T cells resulted in CISH gene KO efficiencies >75%. The CAR transduction efficiency of Vδ1 T cells was not affected by the gene-editing steps incorporated during the generation of the Vδ1 T cells. Compared to CIS^wt CAR Vδ1 T cells, the CISH KO CAR Vδ1 T cell functional activity was associated with improved in vitro expansion and cytolytic activity when co-cultured with B7-H6+ tumor cell lines. In addition, the CISH KO CAR Vδ1 T cells demonstrated prolonged IL-15 mediated JAK-STAT signaling activity, and upregulated transcripts associated with T cell effector activity.

Conclusions

Targeting CIS using CRISPR-Cas gene-editing enhanced the anti-tumor potential of γδ T cells. This approach provides additional insight into further enhancement of allogeneic CAR γδ T cells as a promising platform for cancer immunotherapy.

References

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