CD99 Is a Novel Immunotherapy Target in Diffuse Midline Glioma: Fratricide-Resistant CAR T Cells Enable Durable Tumor Control

Diffuse midline glioma (DMG) is a highly infiltrative brain tumor of the pons and other midline structures with a dismal prognosis. Chimeric antigen receptor (CAR) T cell therapy has shown promise for DMG; however, to date, clinical responses have generally been transient, underscoring the need for strategies that achieve more durable therapeutic benefit. Here, we identify CD99 as a highly and consistently expressed surface antigen in both H3K27M-mutant and histone wild-type DMG, supporting its potential as a pan-DMG therapeutic target. We engineered a second-generation CD99-directed CAR incorporating a single-chain variable fragment targeting a membrane-proximal epitope and a 4-1BB co-stimulatory domain, which demonstrated potent antitumor activity in DMG models. Locoregional delivery resulted in complete tumor regression in orthotopic xenografts. However, endogenous CD99 expression on T cells triggered severe fratricide, limiting expansion and persistence. To overcome this barrier, we generated fratricide-resistant CD99 CAR T cells via CRISPR/Cas9 deletion of CD99, which markedly improved T cell viability and durability, resulting in sustained tumor clearance in aggressive DMG models. Together, these findings establish CD99 as a promising immunotherapeutic target and show that underscoring the target-dependent need for genetic fratricide prevention.