CAR NKT Cell Immunotherapy of Neuroblastoma

摘要 尽管在治疗B细胞恶性肿瘤方面取得了显著进展，但表达嵌合抗原受体的T细胞 (CARS)对实体肿瘤基本上仍然无效。我们证明了与传统的T细胞和CAR-T细胞不同， V-24不变自然杀伤T细胞(NKT)和CAR-NKT在异种模型中有效地向肿瘤部位转运 神经母细胞瘤(NB)，这导致了人类第一个评估自体GD2-1临床试验的启动。 神经营养不良儿童的特异性CAR-NKT。这项试验的中期结果表明，治疗效果良好- 耐受性和产生抗肿瘤活性，强调需要系统地研究NKT作为一种 用于汽车重定向免疫治疗的替代细胞平台。我们报告了反复的抗原刺激 NKT和CAR-NKT的缺失导致其效应器样的分化、衰竭和抗肿瘤活性的丧失。我们的 初步数据表明，Wnt信号转导--特别是转录因子LEF1的激活--是必需的 并且足以维持CD62L中央记忆样NKT子集，并且与NKT相关 新陈代谢和机能健康。这些发现为研究其作用和潜在的治疗方法提供了理论基础。 在CAR-NKT中靶向LEF1用于癌症免疫治疗。CAR-NKT的临床前体内研究已经 仅限于异种模型，不允许评估下游先天和 适应性免疫反应。为了解决这一差距，我们开发了协议，以生成和高度扩展 表达针对人和小鼠NB上表达的GD2抗原的小鼠CAR的纯小鼠NKT 细胞。我们还采用了一种同基因NB模型，忠实地概括了临床和病理 儿童高危神经营养不良的特点。我们假设1)LEF1转录活性是 并可在治疗上增强以保持CAR-NKT细胞的新陈代谢适合性、体内持久性和 持久的抗肿瘤活性；以及2)CAR-NKT的抗肿瘤活性依赖于肿瘤细胞的直接靶向，对照 肿瘤相关巨噬细胞、NK细胞的反式激活和肿瘤特异性T细胞的诱导。这些 假说将在以下具体目标中进行检验：1)检查并从治疗角度探索 负责维持人类中央记忆样NKT和CAR-NKT细胞的机制，以及2) 在同基因神经母细胞瘤模型中探讨CAR-NKT介导抗肿瘤活性的机制。我们将使用 遗传功能丧失和功能获得方法研究LEF1在CAR-NKT功能中的作用 人细胞体外实验系统的分化、代谢和抗肿瘤活性 小鼠体内异种神经母细胞瘤模型的建立。探讨CAR-NKT介导抗肿瘤活性的机制 在同基因环境下，我们将对表达GD2-1的小鼠NKT和T细胞进行并列比较。 特定的汽车。拟议中的实验将从机械上剖析天然NKT细胞的贡献 TCR，肿瘤特异性CAR和治疗诱导的先天和获得性免疫反应。结果将会是 告知针对NB和其他类型癌症的下一代CAR-NKT免疫疗法的发展。

Abstract Despite remarkable progress in treating B cell malignancies, T cells expressing chimeric antigen receptors (CARs) remain largely ineffective in solid tumors. We demonstrated that unlike conventional T and CAR-T cells, V24-invariant natural killer T cells (NKTs) and CAR-NKTs effectively traffic to tumor sites in xenogeneic models of neuroblastoma (NB), which led to initiation of a first-in-human phase 1 clinical trial evaluating autologous GD2- specific CAR-NKTs in children with NB. Interim results from this trial have shown that the treatment is well- tolerated and produces antitumor activity, underscoring the need for systematic investigation of NKTs as an alternative cellular platform for CAR-redirected immunotherapy. We reported that repeated antigenic stimulation of NKTs and CAR-NKTs leads to their effector-like differentiation, exhaustion, and loss of antitumor activity. Our preliminary data demonstrate that Wnt signaling—specifically, activation of transcription factor LEF1—is required and sufficient for maintenance of the CD62L+ central memory-like NKT subset and is associated with NKT metabolic and functional fitness. These findings provide rationale for examining the role and potential therapeutic targeting of LEF1 in CAR-NKTs for cancer immunotherapy. Pre-clinical in vivo studies of CAR-NKTs have been limited to xenogeneic models, which do not allow for evaluation of the full spectrum of downstream innate and adaptive immune responses. To address this gap, we have developed protocols to generate and expand highly pure murine NKTs that express a murine CAR targeting the GD2 antigen expressed on human and murine NB cells. We have also adapted a syngeneic NB model that faithfully recapitulates the clinical and pathological characteristics of high-risk NB in children. We hypothesize that 1) LEF1 transcriptional activity is required for and can be therapeutically enhanced to maintain CAR-NKT cell metabolic fitness, in vivo persistence, and durable antitumor activity; and 2) CAR-NKT antitumor activity depends on direct targeting of tumor cells, control of tumor-associated macrophages, transactivation of NK cells and induction of tumor-specific T cells. These hypotheses will be tested in the following specific aims: 1) to examine and therapeutically explore the mechanism responsible for maintenance of human central memory-like NKT and CAR-NKT cells, and 2) to explore the mechanism by which CAR-NKTs mediate antitumor activity in a syngeneic NB model. We will use genetic loss-of-function and gain-of-function approaches to study the role of LEF1 in CAR-NKT functional differentiation, metabolism, and antitumor activity using in vitro experimental systems with human cells and in vivo xenogeneic NB models in mice. To study the mechanism by which CAR-NKTs mediate antitumor activity in the syngeneic setting, we will perform a side-by-side comparison of murine NKTs and T cells expressing a GD2- specifc CAR. The proposed experiments will mechanistically dissect the contributions of the native NKT cell TCR, the tumor-specific CAR, and therapy-induced innate and adaptive immune responses. The results will inform development of next-generation CAR-NKT immunotherapy for NB and other types of cancer.