Improving CAR T cell therapy for brain tumors using immune competent models

项目总结/摘要 本研究的总体目标是设计一种有效的CAR T细胞疗法用于高级别胶质瘤（HGG）， 这是一个预后不佳的脑肿瘤亚组。CAR T细胞疗法是一项创新技术 基于抗原特异性T细胞的过继转移，所述抗原特异性T细胞被工程化以引起临床有效和特异性的免疫应答。 对肿瘤细胞的免疫反应。HGG患者的早期临床研究证明了CAR T细胞的安全性 脑肿瘤的治疗;然而，仅观察到有限的益处。缺乏疗效很可能是多因素造成的 包括异质性抗原表达、有限的肿瘤归巢和穿透、T细胞耗竭 和有限的持久性，以及免疫抑制肿瘤微环境（TME）。因此 该建议的中心假设是CAR的优化设计和额外的遗传修饰 T细胞将提高其抗肿瘤作用，这些干预措施将在免疫系统中进行评估。 非常接近人类疾病的小鼠模型。提出了三个相互关联的研究目标 以检验这一假设，每个假设的基本原理概述如下。首先，CAR设计必须优化， 在免疫活性胶质瘤模型中进行评估。在目标1中，我将产生含有不同共- 刺激结构域（CD28.mu、41BB.mu、CD28.mut和41BB.mut），并比较它们在体外和体内的活性。 vivo.第二，在原代T细胞中的全基因组筛选已经鉴定了T细胞活化后T细胞活化的关键调节因子。 细胞受体（TCR）刺激。已鉴定的基因属于调节细胞周期、增殖和增殖的分子， 下游TCR信号传导包括SOCS 1、RASA 2或CBLB。因此，在目标2中，我将探讨CRISPR- Cas9介导的Socs 1、Rasa 2和/或Cblb沉默增强CAR T细胞的效应子功能。的 然后，独立的Aim 3将探索双重CAR靶向方法，其中我不仅靶向胶质瘤细胞， 还包括神经胶质瘤微环境中的免疫抑制细胞。这些研究集中在肿瘤 相关巨噬细胞（TAM），因为它们大量存在于胶质瘤中，并在形成胶质瘤中发挥关键作用。 的TME。为了支持这个项目的可行性，我已经适应了完善的免疫能力GL 261 神经胶质瘤模型研究靶向相关肿瘤抗原B7-H3的CAR T细胞疗法，这不仅是 它不仅在GL 261细胞中表达，而且在广泛的儿童和成人脑肿瘤中表达。另外我 初步研究表明，“原型”B7- H3 CAR T细胞在体外容易识别并杀死GL 261细胞 并具有体内抗肿瘤活性，突出表明所开发的模型非常适合于所提出的目标， 这个项目在所有三个目的中将使用最先进的技术，不仅研究功能和体内命运， CAR T细胞，以及它们的抗肿瘤活性，以及CAR T细胞如何与神经胶质瘤浸润性免疫细胞相互作用。 细胞这项研究的完成将确定最佳控制HGG肿瘤的最佳CAR设计， 在炎性脑肿瘤中持续时间更长。此外，结果将说明，如果靶向TAM 将克服TME对B7-H3 CAR T细胞的抑制作用。

PROJECT SUMMARY/ABSTRACT The overall objective of this study is to engineer a potent CAR T cell therapy for high grade glioma (HGG), a subgroup of brain tumors for which outcomes remain poor. CAR T cell therapy is an innovative technology based on adoptive transfer of antigen-specific T cells engineered to elicit a clinically effective and specific immune response against tumor cells. Early clinical studies in HGG patients demonstrated safety of CAR T cell therapy for brain tumors; yet, only limited benefits were observed. Lack of efficacy is most likely multifactorial and include heterogenous antigen expression, limited homing to and penetration of tumors, T cell exhaustion and limited persistence, as well as the immunosuppressive tumor microenvironment (TME). Therefore, the central hypothesis of this proposal is that optimized design and additional genetic modifications of CAR T cells will improve their antitumor effects, and that these interventions will be evaluated in immune competent mouse models that closely mimic human disease. Three interrelated research aims are proposed to test this hypothesis and the rationale of each is outlined below. First, CAR design has to be optimized and evaluated in immune competent glioma models. In Aim 1, I will generate CAR T cells containing different co- stimulatory domains (CD28.ζ, 41BB.ζ, CD28.mutζ, and 41BB.mutζ), and compare their activity in vitro and in vivo. Second, a genome wide screen in primary T cells has identified key regulators of T cell activation post T- cell receptor (TCR) stimulation. Identified genes belong to molecules that regulate cell cycle, proliferation, and downstream TCR signaling including SOCS1, RASA2, or CBLB. In Aim 2, I will therefore explore if CRISPR- Cas9 mediated silencing of Socs1, Rasa2, and/or Cblb enhances the effector functions of CAR T cells. The independent Aim 3 will then explore a dual CAR targeting approach in which I will not only target glioma cells but also immunosuppressive cells within the glioma microenvironment. These studies are focused on tumor associated macrophages (TAMs), since they are abundantly present in gliomas and play a critical role in shaping the TME. To support the feasibility of this project, I have adapted the well-established immune competent GL261 glioma model to study CAR T cell therapies targeting the relevant tumor antigen B7-H3, which is not only expressed by GL261 cells but also in a broad range of pediatric and adult brain tumors. In addition, my preliminary studies indicate that `prototype' B7- H3 CAR T-cells readily recognize and kill GL261 cells in vitro and have antitumor activity in vivo, highlighting that the developed model is well suited for the proposed aims of this project. State of the art technique will be used in all three Aims to not only study the function and in vivo fate of CAR T-cells, but also their antitumor activity, and how CAR T-cells interact with glioma- infiltrating immune cells. Completion of this study will define the most optimal CAR design that best controls HGG tumors and persists longer in the context of inflammatory brain tumors. Additionally, results will illustrate if targeting TAMs will overcome the suppressive effects of TME on B7-H3 CAR T cells.