BCL11B activation as an approach for enhancing the efficacy of immunotherapy

我们的目标是研究T系转录因子(TF)BCL11B的过表达作为一种新的策略 促进：1)异基因造血干细胞移植(HSCT)后T细胞重建； 抗癌嵌合抗原受体(CAR)T细胞的疗效造血干细胞移植是许多白血病的根治疗法。 或者作为一种汽车后巩固疗法。然而，捐献者的T细胞的产生 干细胞和祖细胞(HSPC)需要数月时间才能造成危及生命的感染和白血病复发 HSCT面临的主要挑战。虽然CAR T细胞在CD19+白血病中诱导高缓解率，但贫乏的T细胞 由于肿瘤微环境的抑制，功能和持久性以及T细胞耗尽仍然是主要的 CAR T细胞在白血病和实体瘤中疗效的障碍。与物种相关的差异 转铁蛋白对T细胞分化的调控及对T细胞机制的认识不足 分化一直是发展促进T细胞分化和 功能。肿瘤抑制因子Tf Bcl11b是抑制替代(非T)谱系潜能所必需的 但在T细胞的初始阶段并不起到诱导T系基因表达的作用 小鼠HSPC的分化。相反，我们证明了BCL11B对T细胞的诱导是关键的。 人类T细胞初始阶段的谱系程序和选择性谱系程序的抑制 差异化。我们现在有了新的体外初步数据，慢病毒BCL11B过表达：1)加速 人HSPC的T细胞分化包括成熟T细胞的产生，以及2)增强功能， 促进分化为具有中央记忆表型的细胞，并延缓人类T细胞的耗竭。 对功能、芯片序列和单细胞RNA-序列数据的综合分析表明，NOTCH3和IRF8是 人类BCL11B的物种特异性候选靶点。值得注意的是，BCL11B过度表达的研究并没有 由于毒性，在小鼠HSPC中是可能的。根据这些数据，我们假设HSPC移植 基因工程过表达BCL11B将增强HSCT后T细胞的重建。BCL11B过度表达将 通过增强CAR T细胞的功能和持久性以及改善疲惫来提高其疗效。 我们将通过以下目标来检验这一假说：1.1)确定BCL11B对T细胞的表观遗传效应。 细胞基因及BCL11B介导的NOTCH3(1.2)和IRF8(1.3)对人T细胞的调控作用 差异化。1.4)确定高表达人HSPC的BCL11B对增强人HSPC基因表达的作用。 HSCT T细胞在人源化小鼠模型中的重建，以及2)确定BCL11B过表达的影响 人CAR T细胞在白血病和神经母细胞瘤中的抗癌效果、持久性和耗竭的研究 模特们。这些研究可能揭示BCL11B的新功能，并导致BCL11B工程细胞疗法 可以改善白血病和实体瘤的治疗结果。这项建议是创新的，因为它建立在我们工作的基础上 确定BCL11B在人类中的物种特异性效应，以解决HSCT和CAR T细胞治疗中的关键障碍。

Our goal is to investigate overexpression of the T-lineage transcription factor (TF) BCL11B as a novel strategy to enhance: 1) T-cell reconstitution after allogeneic hematopoietic stem cell transplantation (HSCT), and 2) the efficacy of anticancer chimeric antigen receptor (CAR) T-cells. HSCT is a curative therapy for many leukemias by itself or as a post-CAR consolidation therapy. However, the generation of T-cells from donor hematopoietic stem and progenitor cells (HSPC) takes many months making life threatening infections and leukemia relapse major challenges in HSCT. While CAR T-cells induce high remission rates in CD19+ leukemias, poor T-cell function and persistence and T-cell exhaustion due to inhibition by the tumor microenvironment remain major obstacles to the curative efficacy of CAR T-cells in leukemia and solid tumors. Species related differences in the regulation of T-cell differentiation by TF and the poor understanding of mechanisms in human T-cell differentiation have been hurdles to the development of approaches to enhance T-cell differentiation and function. The tumor suppressor TF Bcl11b is required for the repression of alternative (non-T) lineage potentials but does not play a role in the induction of T-lineage gene expression during the initial stages of T-cell differentiation of murine HSPC. In contrast, we showed that BCL11B is critical for both the induction of the T- lineage program and repression of alternative lineage programs during the initial stages of human T-cell differentiation. We now have novel preliminary in vitro data that lentiviral BCL11B overexpression: 1) expedites T-cell differentiation from human HSPC including the generation of mature T-cells, and 2) enhances the function, promotes differentiation into cells with a central memory phenotype, and delays exhaustion of human T-cells. Integrated analysis of functional, Chip-Seq, and single cell RNA-Seq data revealed NOTCH3 and IRF8 as species specific candidate targets of BCL11B in humans. Of note, BCL11B overexpression studies have not been possible in murine HSPC due to toxicity. Based on these data, we hypothesize that transplantation of HSPC engineered to overexpress BCL11B will enhance post-HSCT T-cell reconstitution. BCL11B overexpression will increase the efficacy of CAR T-cells by enhancing their function and persistence and ameliorating exhaustion. We will test the hypothesis through the following aims: 1.1) Determine the epigenetic effects of BCL11B on T- cell genes and the role of BCL11B mediated regulation of NOTCH3 (1.2) and IRF8 (1.3) in human T-cell differentiation. 1.4) Define the efficacy of BCL11B overexpressing human HSPC for the enhancement of post- HSCT T-cell reconstitution in humanized mouse models, and 2) Define the effects of BCL11B overexpression on anti-cancer efficacy, persistence, and exhaustion of human CAR T-cells in leukemia and neuroblastoma models. These studies could reveal new functions of BCL11B and lead to BCL11B engineered cell therapies that improve outcomes in leukemia and solid tumors. This proposal is innovative because it builds on our work defining species specific effects of BCL11B in humans to address key barriers in HSCT and CAR T-cell therapy.