Regnase-1 Disruption Drives a CD4-Supported Therapeutic State that Enhances CAR T-Cell Efficacy against Pediatric Brain Tumor

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Abstract
B7‑H3 is a promising immunotherapeutic target for pediatric brain tumors, yet the durability of B7‑H3-CAR T cell responses remains limited. Here, for the first time, we systematically evaluated whether knockout (KO) of TET2, DNMT3A, or Regnase‑1 (REG-1) enhances the antitumor activity of B7‑H3–CAR T cells. Across multiple G3MB orthotopic xenograft models spanning low to high antigen density, second‑generation B7‑H3–CAR T cells improved survival but failed to achieve curative responses, with efficacy influenced by donor‑specific variability and antigen density. While DNMT3A-KO enhanced CAR T cell persistence in vitro, neither TET2 nor DNMT3A-KO improved tumor control in vivo. In contrast, REG‑1-KO significantly augmented antitumor activity, inducing complete tumor eradication in a subset of mice and extending survival across both potent and poor‑performing donors. Single-cell transcriptomic and functional analyses show increased cytokine production, coordinated CD4–CD8 communication, and reduced exhaustion-associated programs, supporting a model in which effective responses depend on CD4-mediated immune orchestration rather than persistence or cytotoxicity alone. We also demonstrate CAR specificity: while DNMT3A and Regnase-1 disruption both enhance EphA2-targeted CAR T cells, only Regnase-1 disruption improves B7-H3 CAR T-cell efficacy, underscoring the need for context-specific engineering. Our findings challenge the prevailing assumption that persistence-enhancing strategies are central to successful CAR T cell therapies. Instead, they demonstrate that distinct engineering approaches should be considered for the specific disease and CAR target.