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 CISwt 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.
Zhu H, Blum RH, Bernareggi D, Ask EH, Wu Z, Hoel HJ, Meng Z, Wu C, Guan KL, Malmberg KJ, Kaufman DS. Metabolic Reprograming via Deletion of CISH in Human iPSC-Derived NK Cells Promotes In Vivo Persistence and Enhances Anti-tumor Activity. Cell Stem Cell. 2020 Aug 6;27(2):224–237.
Palmer DC, Guittard GC, Franco Z, Crompton JG, Eil RL, Patel SJ, Ji Y, Van Panhuys N, Klebanoff CA, Sukumar M, Clever D, Chichura A, Roychoudhuri R, Varma R, Wang E, Gattinoni L, Marincola FM, Balagopalan L, Samelson LE, Restifo NP. Cish actively silences TCR signaling in CD8+ T cells to maintain tumor tolerance. J Exp Med. 2015 Nov 16;212(12):2095–113.
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