A Multi-Kinase Inhibitor Screen Identifies Inhibitors Preserving Stem-Cell-Like Chimeric Antigen Receptor T Cells [ATAC-Seq]
Chimeric antigen receptor T cells (CAR-Ts) with T-stem cell (TSCM)-like phenotypic characteristics promote sustained antitumor effects. We performed an unbiased and automated high-throughput screen of a kinase-focused compound set to identify kinase inhibitors (KIs) that preserve human TSCM-like CAR-Ts. We identified three KIs UNC10225387B, UNC10225263A and UNC10112761A that combined in vitro increased the frequency of CD45RA+CCR7+TCF1high TSCM-like CAR-Ts from both healthy donors and cancer patients. KIs-treated CAR-Ts showed enhanced antitumor effects both in vitro and in vivo. The KI cocktail maintains TSCM-like phenotype preferentially in CAR-Ts originating from naïve T cells, and promotes dynamic transcriptomic changes without arresting T-cell activation or modulating the chromatin organization. Specific kinases ITK, ADCK3, MAP3K4 and CDK13 targeted by the KI cocktail in a dose-dependent manner are directly associated with the preservation of TSCM-like CAR-Ts. While individual knockdown of these kinases or their combination enriched in TSCM-like CAR-Ts, only CAR-Ts generated in the presence of the KI cocktail show robust expansion and differentiation upon stimulation with tumor cells. Overall, transient pharmacologic inhibition of strategically targeted kinases maintains stem-like features of CAR-Ts and improves their antitumor activity. Overall design: Human T-cells were isolated from collected PBMCs from two healthy donors, maintained with anti-CD3 and anti-CD28 antibodies and transduced with a CD19-directed CAR via retroviral vector prior to expansion using IL-7 and IL-15.  These cells were exposed to either DMSO or a collection of kinase inhibitors (KIs).  Samples were collected prior to exposure (baseline), and at 1, 3 and 7 days post-exposure before being sequenced via RNA and/or ATAC-seq.