Timosaponin AIII enhances CAR-T cell potency and prevents relapse through impairing CAR-Tregs

Chimeric antigen receptor (CAR)-T cell therapy has become the standard treatment for refractory and relapsed (r/r) DLBCL in recent years. However, more than half of these patients develop primary or secondary resistance. Given that CAR Tregs are crucial in driving resistant and relapse to CAR-T therapy, but the strategies to overcome are limited. Here, we established a Treg functional high-throughput screening system and identified a natural product, currently in clinical trials, Timosaponin AIII (TAIII), impairs Treg function while activating T cell immune response during CAR-T therapy. Mechanistically, TAIII recruits and blocks the A2AR signaling in CAR-T cells as a novel allosteric inhibitor of A2AR, thereby eliminating CAR-Tregs by suppressing CREB-mediated FoxP3 transcription. Furthermore, TAIII suppresses tumor growth and improves the efficacy of PD-1 immunotherapy by reducing intratumoral Tregs in solid tumor models. Importantly, TAIII increases the Tcm percentage and enhances cytotoxic cytokine secretion of CAR-T cells post-infusion, improving their function. Combining or pretreating CAR-T cells with TAIII enhances their antitumor activity, thereby overcoming late relapses in lymphoma models. These results identify TAIII as a potent adjuvant for CAR-T cell function and manufacturing through eliminating CAR-Tregs proportion, thereby enabling CAR-T therapy to be applied with a broader perspective.