Abstract 7801: Preclinical efficacy of DLL3 targeted CAR T cells in neuroblastoma

Abstract 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 (p0.05). 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 (p0.001). 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. Citation Format: Salvatore M. Aspromonte, Tamar Feinberg, Ali Cihan, Samantha Brosius, Armaan H. Siddiquee, Dylan Domenico, Daoqi You, Shakeel Modak, Kevin J. Curran, Andrew L. Kung, Anthony Daniyan, Filemon Dela Cruz. Preclinical efficacy of DLL3 targeted CAR T cells in neuroblastoma abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7801.