Michaela M. Meehl

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.

Figure S13 legend1 from B7-H3 CAR T Cells Are Effective against Ependymomas but Limited by Tumor Size and Immune Response

Figure S4 shows antigen profiling of HER2 in ZFTA-fusion driven EPN cell lines.

Figure S16 shows high potency but limited durability of B7-H3.CAR T-cells against ZFTA-RELA EPN xenograft tumors in vivo.

Figure S20 shows in vitro efficacy of murine B7-H3.CAR T-cells against murine ZFTA-RELA EPN cell line.

Figure S19 shows successful infiltration of HER2.CAR T-cells inside 1425 EPN xenograft tumors.

Supplementary Tables S1 and S2

Figure S14 shows comparison of phenotypic characteristics between B7-H3.CAR and HER2.CAR T-cells during repeated stimulation with 1425 cell line.

Figure S15 shows increased inhibitory marker expression on B7-H3.CAR T-cells compared to HER2.CAR T-cells following repeated stimulation with 1425 cell line.

Figure S11 shows phenotypic characteristics of human HER2.CAR T-cells.