ID #458 Tailored gene-editing to improve B7-H3 CAR T-cell therapy for group 3 medulloblastoma

Abstract Medulloblastoma (MB) is the most common malignant pediatric brain tumor with molecular Group 3 (G3MB) having poor prognosis with few recent advancements in treatment. Chimeric antigen receptor (CAR) T-cell therapy targeting the antigen B7-H3 is safe for pediatric brain tumor patients, including G3MB, but has had limited clinical success in eliciting durable responses in part due to poor CAR T-cell fitness. In this work, we sought to determine whether improving the capacity of B7-H3-CAR T-cells to 1) persist or 2) expand against G3MB would improve anti-tumor efficacy in patient derived xenograft (PDX) models. We used CRISPR/Cas9 to generate healthy donor-derived B7-H3-CAR T-cells with a greater potential to persist by knocking out DNMT3A or TET2 or expand by knocking out Regnase-1. As controls, we generated DNMT3A, TET2, and Regnase-1 knock out (KO) EphA2-CAR T-cells. We directly compared these CAR T-cell products against B7-H3+/EphA2+ PDX G3MB cell lines in vivo and found that only Regnase-1 KO improved the anti-tumor activity of B7-H3-CAR T-cells whereas DNMT3A or Regnase-1 KO improved the antitumor activity of EphA2-CAR T-cells. Regnase-1 KO B7-H3-CAR T-cells had a superior capacity to expand and, unexpectedly, maintained a high percentage of CD4+ CAR T-cells compared to all other KOs. Next, we validated the clinical potential of Regnase-1 KO in B7-H3-CAR T-cells generated from T cells isolated from pediatric brain tumor patients from our Loc3CAR clinical trial (NCT05835687). We show that Regnase-1 KO indeed improves the anti-tumor efficacy of patient-derived B7-H3-CAR T-cells in vivo, nominating Regnase-1 KO B7-H3 CAR-T cells as a future CAR T-cell product for clinical testing for pediatric brain tumors. Beyond the translational relevance of our work, our study highlights that the benefit of deleting negative regulators in T cells is CAR-dependent, highlighting the need to delineate underlying mechanisms going forward.