supporting data for ''An Inducible Safety Enhanced TET2-knockdown System for Improving Chimeric Antigen Receptor T Efficacy in Solid Tumors''

Chimeric antigen receptor (CAR) T-cell therapy, as one of the most revolutionary breakthroughs in modern medicine, has transformed the treatment landscape for specific hematologic malignancies. However, it for solid tumors is hindered by poor T-cell persistence and function within the immunosuppressive tumor microenvironment. While constitutive knockout of the epigenetic regulator TET2 enhances CAR-T cell expansion and persistence, it raises significant long-term safety concerns, including antigen-independent systemic tissue infiltration and malignant transformation. To harness the benefits of TET2 loss while mitigating its risks, we developed a novel TET2 knockdown system with dual safety features: a doxycycline (Dox)-inducible Tet-On system to control TET2-targeting shRNA expression and an embedded CD20 mimotope for rituximab-mediated ablation. Bivalent CARs targeting GD2 and B7-H3 were designed to avoid antigen-escape and were tested against Ewing sarcoma and neuroblastoma cells. We demonstrated that Dox-induced TET2 knockdown significantly enhanced the proliferation of CAR-T cells in vitro and in vivo without altering their cytotoxic function. In the xenograft model, Dox administration profoundly augmented CAR-T cell homing and infiltration into tumors, inducing extensive necrosis. Critically, the TET2 knockdown phenotype was reversible, and the engineered cells could be eliminated through the administration of Rituximab. Thus, this bivalent CAR with inducible TET2-knockdown system offers a new therapeutic strategy for improving CAR-T therapy in solid tumors, providing a balance between enhanced efficacy and controllable safety.