Engineering a smart CAR platform for human regulatory T cells

表达肿瘤细胞上靶向抗原的嵌合抗原受体(CAR)的T淋巴细胞 在抗击癌症方面展示了巨大的希望，去年有两种疗法获得了FDA的批准 年。除了免疫激活外，CARS还可以抑制抗原依赖型患者的免疫反应 在调节性T淋巴细胞(Treg)上表达的方式。汽车树在治疗方面显示出了希望 临床前研究中的自身免疫性疾病、移植物对宿主疾病和移植排斥反应 与目前的治疗方法相比，它具有更多的活性和更少的偏离目标的影响。因此，在安全性和特异性方面的进展 CAR Treg疗法可能会对全球数百万患者产生巨大的有益影响。 汽车Treg疗法面临的一个障碍是，目前在诊所发现的汽车设计只能针对 单一抗原具有有限的特异性，这是Treg治疗中的一个主要问题。此外，这些目前的设计是 过于僵化，不利于优化。为了解决这些问题，我们的实验室最近开发了一种分离式、通用型 和可编程(以上)汽车系统，同时包含多个关键升级。这些 这些特性可以缓解过度激活，并增强工程T细胞的特异性。 随着CAR Treg疗法通过临床试验迅速发展，了解CARS的作用机制 在Tregs中迫切需要促进汽车Treg治疗的进展。然而，我们目前的机制 对汽车如何工作的了解有限，特别是在特雷格汽车上。鉴于蛋白质磷酸化起到了 在CAR信号中的关键作用，对CAR Treg激活的全球磷酸蛋白质组分析将减少我们的 通过发现新的信号通路和确定潜在的优化目标来实现知识差距。 在为这项提案做准备时，我们已经展示了我们的SUPRA CAR系统在 人类原始树。我们还开发了一个定量质谱学工作流程来审问 人类T细胞信号的磷酸蛋白质组学。我们将解决我们的假设，通过一个更好的 了解汽车信号和一个可编程和可感应的汽车系统，我们可以实现更高的 对Treg免疫反应的控制水平，这最终将导致更有效和更安全的治疗 范围广泛的自身免疫性疾病。我们建议将合成生物学和Treg生物学相结合，以扩展 我们在初级树上的CAR系统的能力，并随后使用蛋白质组学来开发一种 对车载系统如何控制Treg信号的机械理解。具体来说，我们的目标是： 目标1：扩展上述CARS中的信号结构域，以控制Treg反应。 目的2：建立人树突状细胞的组合和逻辑抗原检测方法。 目的3：定义Tregs中SUPRA CARS信号的全球磷酸蛋白质组学特征。 这项关于汽车设计的工作可以显著提高Treg疗法的安全性和特异性 补充现有的努力，强调优化Treg活动。

T lymphocytes expressing a chimeric antigen receptor (CAR) targeting antigens on tumor cells have demonstrated tremendous promise in combating cancer, with two therapies receiving FDA-approval in the last year. In addition to immune activation, CARs can suppress immune responses in an antigen-dependent manner when expressed on regulatory T lymphocytes (Tregs). CAR Tregs have shown promise in treating autoimmune disease, graft vs. host disease, and transplant rejection in preclinical studies with improved activity and fewer off-target effects than current treatments. As such, advances in the safety and specificity of CAR Treg therapy could have a huge beneficial impact to millions of patients worldwide. An obstacle facing CAR Treg therapy is that the current CAR designs found in the clinic can only target a single antigen with limited specificity, a major concern in Treg therapy. Additionally, these current designs are too rigid to facilitate optimization. To address these issues, our lab has recently developed a split, universal and programmable (SUPRA) CAR system that simultaneously encompasses multiple critical upgrades. These features can mitigate over-activation, and enhance specificity of the engineered T cells. As CAR Treg therapies rapidly progress through clinical trials, understanding the mechanism of action of CARs in Tregs is urgently needed to facilitate CAR Treg therapy advancement. However, our current mechanistic understanding of how CARs work, especially in Tregs, is limited. Given that protein phosphorylation plays a critical role in CAR signaling, a global phosphoproteomic analysis on CAR Treg activation will reduce our knowledge gap by uncovering novel signaling pathways and identifying potential targets for optimization. In preparation for this proposal, we have demonstrated that our SUPRA CAR system is fully functional in human primary Tregs. We have also developed a quantitative mass spectrometry workflow to interrogate the phosphoproteomics of human T cell signaling. We will address our hypothesis that through a better understanding of CAR signaling and a programmable and inducible CAR system, we can achieve a higher level of control over Treg immune response, which will ultimately lead to a more effective and safer therapy for a wide range of autoimmune diseases. We propose to combine synthetic biology and Treg biology to expand the capability of our SUPRA CAR system in primary Tregs, and subsequently use proteomics to develop a mechanistic understanding on how the SUPRA CAR system controls Treg signaling. Specifically, we aim to: Aim 1: Expand the signaling domain repertoire in SUPRA CARs to control Treg responses. Aim 2: Establish combinatorial AND logic antigen detection in human Tregs. Aim 3: Define global phosphoproteomics signatures of SUPRA CARs signaling in Tregs. This work on CAR design could significantly improve the safety and specificity of Treg therapies and complement existing efforts that emphasize the optimization of Treg activity.