CRISPR/Cas9-Mediated Knockout of DGK Improves Antitumor Activities of Human T Cells

Abstract

The efficacy of T-cell therapy is inhibited by various tumor-associated immunosuppressive ligands and soluble factors. Such inhibitory signals turn specific T-cell signaling pathways on or off, impeding the anticancer functions of T cells. Many studies have focused on PD-1 or CTLA-4 blockade to invigorate T-cell functions through CD28/B7 signaling, but obtaining robust clinical outcomes remains challenging. In this study, we use CRISPR/Cas9 to potentiate T-cell function by increasing CD3 signaling via knockout of diacylglycerol kinase (DGK), an enzyme that metabolizes diacylglycerol to phosphatidic acid. Knockout of DGK augmented the effector functions of CAR-T cells via increased TCR signaling. DGK knockout from CAR-T cells rendered them resistant to soluble immunosuppressive factors such as TGFβ and prostaglandin E2 and sustained effector functions under conditions of repeated tumor stimulation. Moreover, DGK knockout caused significant regression of U87MGvIII glioblastoma tumors through enhanced effector functions in a xenograft mouse model. Collectively, our study shows that knockout of DGK effectively enhances the effector functions of CAR-T cells, suggesting that CRISPR/Cas9-mediated knockout of DGK could be applicable as part of a multifaceted clinical strategy to treat solid cancers. This novel study demonstrates efficient ablation of diacylglycerol kinase in human CAR-T cells that leads to improved antitumor immunity and may have significant impact in human cancer immunotherapy. .

T-cells “à la CAR-T(e)” – Genetically engineering T-cell response against cancer

“…Indeed, these approaches were demonstrated to reliably knock down the expression of PD1 and other checkpoint inhibitors or key signaling molecules [280][281][282][283] and further optimization has been performed to delineate proper conditions for CRISPR editing in primary T cells [284] showing the successful silencing of several checkpoint targets. Unexpectedly, Fraietta et al recently showed that in the case of a CLL patient that achieved complete remission when treated with anti-CD19 CAR T-cells, the insertion of the CAR transgene caused a disruption in the methylcytosine dioxygenase TET2 gene [285].…”

CRISPR-Cas, a robust gene-editing technology in the era of modern cancer immunotherapy

“…Furthermore, immune checkpoint blockade of inhibitory receptors (PD-1, Tim-3, CTLA-4, LAG-3, DGK, FAS, etc.) postpones CAR T-cells exhaustion and reinforces them for clinical trials [ 99 ]. Both HLA knockout and immunosuppressive pathways blockade can be achieved by means of CRISPR without any detrimental immunological side-effects as observed during employment of blocking antibodies (Fig.…”

Immunotherapy in Glioblastoma: Current Shortcomings and Future Perspectives

“…The knock-out of the intracellular signaling molecule diacylglycerol kinase (DGK) using a CRISPR/Cas9-based strategy in EGFRvIII CAR T cells led to a significantly less sensitivity to TGF-β mediated suppression and did not show significant loss of effector function following repeated stimulation. These in vitro findings translated to in vivo studies in a mouse glioma model as tumor-bearing mice receiving double knock-out EGFRvIII CAR T cells had significantly reduced tumor burden with increased frequencies of tumor-infiltrating lymphocytes [93]. The-knock in of genes to enhance CAR T cell function has not yet been tested in glioblastoma [91].…”