NR4a knock-down in HER2 CAR-T cells improves T cell longevity and promotes solid tumor eradication [RNA-Seq]

Chimeric antigen receptor (CAR)-T cell therapy targeting human CD19 have demonstrated clinical efficacy against B-cell malignancies. However, CAR-T cell therapy's efficacy against solid tumors is limited due to factors like low tumor-associated antigens, infiltration rate, and T cell exhaustion. We have shown that deleting NR4a genes in CAR-T cells prevents T cell exhaustion and improved their therapeutic effects on solid tumors in a mouse model. To further explore this for human, we deleted all three NR4a family factors in CAR-T cells that recognize Epidermal Growth Factor Receptor type 2 (HER2) using the CRISPR/Cas9 system. These modified CAR-T cells (NR4a-TKO CAR-T) exhibited resistance to exhaustion, increased tumor-killing activity, and higher efficacy in tumor regression and survival rate in a human lung carcinoma model in mice. The enhanced therapeutic effects were associated with increased cytokine expression, reduced exhaustion-related gene expression, and improved persistence within tumors. We propose that targeting NR4a could be a promising strategy for developing superior CAR-T cells against solid tumors. Overall design: Primary human T cells were isolated from PBMC derived from healthy donors and activated with anti-CD3/CD28 Dynabeads in AIM-V medium supplemented with IL-2 (10ng/mL). After 24 hours, the anti-HER2 CAR-Transgene was transduced with a Lentivirus into the T cells using RetroNectin (Takara Bio). On day 3, Dynabeads were magnetically removed, and CAR-T cells were electroporated with Cas9-RNPs.