Engineering a smart CAR platform for human regulatory T cells

表达靶向肿瘤细胞上的抗原的嵌合抗原受体（CAR）的T淋巴细胞， 在对抗癌症方面表现出了巨大的希望，去年有两种疗法获得了FDA的批准。 年除了免疫激活之外，汽车还可以以抗原依赖性免疫应答抑制免疫应答。 在调节性T淋巴细胞（Tlymphocyte，Tlymphocyte）上表达的方式。CAR Tandem显示出治疗 自身免疫性疾病、移植物抗宿主病和移植排斥的临床前研究， 活性和更少的脱靶效应比目前的治疗。因此，在安全性和特异性方面的进展 CAR Treg疗法可能对全球数百万患者产生巨大的有益影响。 CAR Treg疗法面临的障碍是，目前在临床中发现的CAR设计只能靶向一种免疫调节因子。 具有有限特异性的单一抗原，这是Treg治疗中的主要问题。此外，这些现有设计 过于死板，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裂的通用 和可编程（SUPRA）CAR系统，同时包含多个关键升级。这些 这些特征可以减轻过度活化，并增强工程化T细胞的特异性。 随着CAR Treg疗法在临床试验中的快速进展，了解汽车的作用机制， 目前迫切需要在Tumor中进行研究，以促进CAR Treg治疗的进展。然而，我们目前的机械 对汽车如何工作的理解，特别是在Tibetan，是有限的。鉴于蛋白质磷酸化在 在CAR信号传导中的关键作用，对CAR Treg激活的全球磷酸化蛋白质组学分析将减少我们的研究。 通过发现新的信号通路和识别潜在的优化目标，我们可以填补知识空白。 在准备这项提案时，我们已经证明了我们的SUPRA CAR系统在以下方面功能齐全： 人原发性甲状腺肿我们还开发了一种定量质谱工作流程， 人类T细胞信号传导的磷酸蛋白质组学。我们将讨论我们的假设，通过更好的 理解CAR信号传导和可编程和诱导的CAR系统，我们可以实现更高的 控制Treg免疫应答的水平，这将最终导致更有效和更安全的治疗。 一系列自身免疫性疾病我们建议将联合收割机合成生物学和Treg生物学相结合， 我们的SUPRA CAR系统在原发性甲状腺肿中的能力，随后使用蛋白质组学来开发一种 对SUPRA CAR系统如何控制Treg信号传导的机制理解。具体而言，我们的目标是： 目的1：扩展SUPRA汽车中的信号传导结构域库以控制Treg应答。 目的2：建立人T淋巴细胞抗原的组合AND逻辑检测方法. 目的3：定义THEX中SUPRA汽车信号传导的整体磷酸化蛋白质组学特征。 这项CAR设计工作可以显著提高Treg疗法的安全性和特异性， 补充强调Treg活性优化的现有努力。

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.