PO.CL06.01 · 临床研究

DLL3靶向CAR-T细胞在神经母细胞瘤中的临床前疗效

Preclinical efficacy of DLL3 targeted CAR T cells in neuroblastoma

编号 7801 展板 4 时间 4/22 09:00–12:00 区域 Section 44 主讲 Salvatore Aspromonte, BS;MD

分会场 Immunotherapies in Pediatric Cancers

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作者与单位 Authors & Affiliations

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

Memorial Sloan Kettering Cancer Center, New York, NY

摘要 Abstract

中文摘要

背景：神经母细胞瘤（NBL）是儿童最常见的颅外实体瘤，占儿童癌症死亡的15%，复发后的生存率仍<50%。为扩展免疫治疗选择，我们评估了Delta样配体3（DLL3），这是一种抑制性NOTCH配体，在健康组织中通常被沉默，但在神经内分泌肿瘤中异常表达。NBL中的NOTCH失调以及提示DLL3表达的新兴数据集，支持将DLL3作为靶点进行研究，并评估最初为小细胞肺癌开发的IL-18装甲型DLL3靶向CAR-T细胞。方法：通过RNA测序、免疫组化（IHC）和流式细胞术评估DLL3在NBL患者样本和PDX模型中的表达。在多个效应细胞与肿瘤细胞（E:T）比例下，对IL-18装甲型DLL3 CAR-T细胞体外抗NBL细胞系进行了测试。使用Vardi检验进行曲线下面积分析比较累积杀伤，并采用Wilcoxon秩和检验评估各个E:T比例下的差异。结果：对MSK治疗的儿童实体瘤（n=540）进行RNA测序显示，NBL（n=109）相较于其他非中枢神经系统肿瘤具有最高的DLL3转录本水平（0-19.7 TPM；中位数0.38）。在NBL患者样本中，IHC（n=31）显示52%（16/31）存在DLL3蛋白表达（H评分≥10，范围10-300，中位数100），且转录本与蛋白具有强一致性（Spearman r=0.847，p=2×10⁻⁹）。在NBL PDX模型中，63%（19/30）检测到DLL3蛋白表达，H评分范围10-123（中位数73）。对涵盖H评分7-105的五个PDX模型进行流式细胞术显示，25-88%的肿瘤细胞表面表达DLL3，证实了抗原的可及性。IL-18装甲型DLL3-CAR-T细胞对两种DLL3+ NBL细胞系SK-N-DZ（51% DLL3+，p=0.03）和SK-N-SH（29% DLL3+，p=0.03）的累积细胞毒性显著高于未转导T细胞，并在多个E:T比例下均有活性（p<0.05）。为确认抗原特异性，将IL-18装甲型DLL3-CAR-T细胞与临床级IL-18装甲型CD371靶向CAR（CLEAR AML）作为非DLL3靶向对照进行比较。DLL3-CAR-T细胞对两种细胞系的细胞毒性显著更强（SK-N-DZ，p=0.0006；SK-N-SH，p=0.0004），并在所有E:T比例下均优于对照CAR（p<0.001）。结论：DLL3在部分NBL患者肿瘤和PDX模型中表达，且可用于CAR靶向。IL-18装甲型DLL3靶向CAR-T细胞对DLL3+ NBL模型显示出强大的体外细胞毒性。在DLL3+ NBL PDX中的体内测试正在进行中。这些发现支持DLL3作为一个有前景的免疫治疗靶点，并为进一步开发针对NBL的DLL3靶向CAR-T疗法提供了依据。

查看英文原文 English 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 (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.

利益披露 Disclosure

S. M. Aspromonte, None.. T. Feinberg, None.. A. Cihan, None.. S. Brosius, None.. A. H. Siddiquee, None.. D. Domenico, None.. D. You, None.. S. Modak, None. K. J. Curran, Novartis Independent Contractor, ). Turn Bio g., Board of Directors, non-salaried role), Stock. PromiCell g., Board of Directors, non-salaried role), Stock. Cellectis ). Atara Bio ). A. L. Kung, Karyopharm Therapeutics g., Board of Directors, non-salaried role). DarwinHealth g., Board of Directors, non-salaried role). Isabl g., Board of Directors, non-salaried role), Stock, Patent. Labcorp Other Intellectual Property. A. Daniyan, Biolumina Independent Contractor. Caribou Biosciences, Inc Patent. Hierax Therapeutics, Inc. Independent Contractor. NomoCan Pharmaceuticals LLC Independent Contractor. PromiCell Therapeutics, Inc. g., Board of Directors, non-salaried role), Stock, Patent. Shoreline Biosciences, Inc. Independent Contractor. Syndax Patent. Tigen Pharma SA Patent. F. Dela Cruz, Eisai ). Y-mAbs Therapeutics ).

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