EBAG9 silencing exerts an immune checkpoint function without aggravating adverse effects

Abstract

Chimeric antigen receptor (CAR) Tcells have revolutionized treatment of B cell malignancies. However, enhancing the efficacy of engineered Tcells without compromising their safety is warranted. The estrogen receptor-binding fragment-associated antigen 9 (EBAG9) inhibits release of cytolytic enzymes from cytotoxic T lymphocytes. Here, we examined the potency of EBAG9 silencing for the improvement of adoptive Tcell therapy. MicroRNA (miRNA)-mediated EBAG9 downregulation in transplanted cytolytic CD8+ Tcells (CTLs) from immunized mice improved their cytolytic competence in a tumor model. In tolerant female recipient mice that received organ transplants, a minor histocompatibility antigen was turned into a rejection antigen by Ebag9 deletion, indicating an immune checkpoint function for EBAG9. Considerably fewer EBAG9-silenced human CAR Tcells were needed for tumor growth control in a xenotransplantation model. Transcriptome profiling did not reveal additional risks regarding genotoxicity or aberrant differentiation. A single-step retrovirus transduction process links CAR or TCR expression with miRNA-mediated EBAG9 downregulation. Despite higher cytolytic efficacy, release of cytokines associated with cytokine release syndrome remains unaffected. Collectively, EBAG9 silencing enhances effector capacity of TCR- and CAR-engineered Tcells, results in improved tumor eradication, facilitates efficient manufacturing, and decreases the therapeutic dose.