Runx3-overexpression cooperates with ex vivo AKT inhibition to generate receptor-engineered T cells with better persistence, tumor-residency, and antitumor ability
Jianghui Tang, Jianpeng Sheng, Qi Zhang, Yongtao Ji, Xun Wang, Junlei Zhang, Jiangchao Wu, Jinyuan Song, Xueli Bai, Tingbo Liang
Abstract
Background Solid tumors pose unique roadblocks to treatment with chimeric antigen receptor (CAR) T cells, including limited T-cell persistence, inefficient tumor infiltration, and an immunosuppressive tumor microenvironment. To date, attempts to overcome these roadblocks have been unsatisfactory. Herein, we reported a strategy of combining Runx3 (encoding RUNX family transcription factor 3)-overexpression with ex vivo protein kinase B (AKT) inhibition to generate CAR-T cells with both central memory and tissue-resident memory characteristics to overcome these roadblocks. Methods We generated second-generation murine CAR-T cells expressing a CAR against human carbonic anhydrase 9 together with Runx3 -overexpression and expanded them in the presence of AKTi-1/2, a selective and reversible inhibitor of AKT1/AKT2. We explored the influence of AKT inhibition (AKTi), Runx3 -overexpression, and their combination on CAR-T cell phenotypes using flow cytometry, transcriptome profiling, and mass cytometry. The persistence, tumor-infiltration, and antitumor efficacy of CAR-T cells were evaluated in subcutaneous pancreatic ductal adenocarcinoma (PDAC) tumor models. Results AKTi generated a CD62L+central memory-like CAR-T cell population with enhanced persistence, but promotable cytotoxic potential. Runx 3-overexpression cooperated with AKTi to generate CAR-T cells with both central memory and tissue-resident memory characteristics. Runx3 -overexpression enhanced the potential of CD4+CAR T cells and cooperated with AKTi to inhibit the terminal differentiation of CD8+CAR T cells induced by tonic signaling. While AKTi promoted CAR-T cell central memory phenotype with prominently enhanced expansion ability, Runx3 -overexpression promoted the CAR-T cell tissue-resident memory phenotype and further enhanced persistence, effector function, and tumor-residency. These novel AKTi-generated Runx3 -overexpressing CAR-T cells exhibited robust antitumor activity and responded well to programmed cell death 1 blockade in subcutaneous PDAC tumor models. Conclusions Runx3 -overexpression cooperated with ex vivo AKTi to generate CAR-T cells with both tissue-resident and central memory characteristics, which equipped CAR-T cells with better persistence, cytotoxic potential, and tumor-residency ability to overcome roadblocks in the treatment of solid tumors.