Preclinical efficacy of DLL3 targeted CAR T cells in neuroblastoma

Background: Neuroblastoma (NBL), the most common extracranial solid tumor in children, accounts for 15% of pediatric cancer deaths, and survival after relapse remains <50%. To expand immunotherapeutic options, we evaluated Delta-like ligand 3 (DLL3), an inhibitory NOTCH ligand normally silenced in healthy tissues but aberrantly expressed in neuroendocrine tumors. NOTCH dysregulation in NBL and emerging datasets indicating DLL3 expression support investigation of DLL3 as a target and assessment of IL-18-armored DLL3-directed CAR T cells originally developed for small-cell lung cancer. Methods: DLL3 expression was assessed in NBL patient samples and PDX models by RNA sequencing, immunohistochemistry (IHC), and flow cytometry. IL-18-armored DLL3 CAR T cells were tested in vitro against NBL cell lines across multiple effector-to-tumor (E:T) ratios. Cumulative killing was compared using Vardi's test for area-under-the-curve analysis, and Wilcoxon rank-sum tests evaluated differences at individual E:T ratios. Results: RNA sequencing of pediatric solid tumors treated at MSK (n=540) showed that NBL (n=109) had the highest DLL3 transcript levels relative to other non-CNS tumors (0-19.7 TPM; median 0.38). In NBL patient samples, IHC (n=31) demonstrated DLL3 protein expression (H-score ≥10, range 10-300, median 100) in 52% (16/31) with strong transcript-protein concordance (Spearman r=0.847, p=2×10 -9 ). In NBL PDX models, DLL3 protein expression was detected in 63% (19/30), with H-scores ranging 10-123 (median 73). Flow cytometry across five PDX models spanning H-scores 7-105 showed DLL3 surface expression on 25-88% of tumor cells, confirming antigen accessibility. IL-18-armored DLL3-CAR T cells exhibited significantly greater cumulative cytotoxicity than untransduced T cells against two DLL3+ NBL cell lines, SK-N-DZ (51% DLL3+, p=0.03) and SK-N-SH (29% DLL3+, p=0.03), with activity across multiple E:T ratios (p<0.05). To confirm antigen specificity, IL-18-armored DLL3-CAR T cells were compared with a clinical-grade IL-18-armored CD371-directed CAR (CLEAR AML) as a non-DLL3-targeting control. DLL3-CAR T cells exhibited significantly greater cytotoxicity against both cell lines (SK-N-DZ, p=0.0006; SK-N-SH, p=0.0004) and outperformed the control CAR across all E:T ratios (p<0.001). Conclusions: DLL3 is expressed in a subset of NBL patient tumors and PDX models and is accessible for CAR targeting. IL-18-armored DLL3-directed CAR T cells show strong in vitro cytotoxicity against DLL3+ NBL models. In vivo testing in a DLL3+ NBL PDX is underway. These findings support DLL3 as a promising immunotherapeutic target and justify further development of DLL3-directed CAR T therapy for NBL.