Interrogating and rewiring cell signaling pathways in CAR-T cells with synthetic phosphotyrosine recognition domains

项目摘要 在治疗血液系统恶性肿瘤的最新突破中，嵌合抗原受体(CAR)-T 细胞治疗是肿瘤免疫治疗中最有前途的进展之一。然而，CAR-T细胞 对实体瘤的治疗效果有限。一个主要的障碍是免疫抑制肿瘤。 促进T细胞耗竭的实体肿瘤微环境。另一个挑战是对 CAR结构如何影响驱动T细胞功能的信号机制，这阻止了 合理设计一种具有强大的抗肿瘤活性和抗疲劳的高级CAR疗法。 我是加州大学旧金山分校的达蒙·鲁尼恩癌症研究博士后研究员。我的导师是詹姆斯·威尔斯博士，一位 我是化学生物学和蛋白质工程方面的专家，我的共同导师是T细胞专家Arthur Weiss博士 生物学。酪氨酸磷酸化是CAR-T细胞信号通路的中心组成部分，但 可用于研究Py修饰的工具非常有限。在我的博士后培训期间，我培养了一种 创新和可推广的平台，称为“Py-靶向重组抗体对”或“Py-Trap”，以 设计高度特异和紧密的Py结合域。这一革命性的工具是第一个用于 在三维蛋白质结构中防止Py修饰的工程特定结合剂。它提供了一个 开发策略的平台，用于设计和重新连接CAR-T细胞中由Py介导的信号通路。 在这项工作中，我建议开发基于Py-Trap的策略来应对CAR-T细胞的关键挑战 治疗。在目标1中，我将使用一种新颖的方法来确定不同的汽车结构是如何调节汽车相互作用的 依赖Py的邻近结扎法。这项工作将揭示汽车信号体的关键区别 不同的汽车设计，并揭示了它们功能差异背后的机制。在目标2中，我将确立 一种筛选方法，以确定变化的CAR结构如何影响细胞对PD-1信号的反应。 这项工作将进一步揭示如何合理设计具有增强抵抗力的CAR-T细胞 免疫抑制。在目标3中，我将在CAR-T细胞中设计一条合成PD-1途径，旨在中和 该通路原有的免疫抑制功能1。这是一种与 现有的策略使CAR-T细胞对TME相关的免疫抑制信号更具抵抗力。已被占用 综上所述，这项提案中概述的研究将有助于加深对CAR-T细胞生物学的理解， 提供知识，为未来的汽车设计提供信息，并展示新的战略，以进行设计和优化 细胞内信号转导结果，以推动细胞疗法的发展。 在我的导师、合作者、顾问和加州大学旧金山分校良好的研究环境的支持下，我 将接受蛋白质组学、定量数据分析和T细胞生物学方面的培训。这些技能将帮助我达到 我的长期目标是成为一家进行严格科学研究调查的实验室的负责人 设计人类免疫系统的新策略。 1目标3包含专有/特权信息，新周博士要求不得向政府以外的人发布这些信息，但出于以下目的 评审和评估。

Project Summary Among recent breakthroughs in treating hematopoietic malignancies, chimeric antigen receptor (CAR)-T cell therapy is one of the most promising advances in cancer immunotherapy. However, CAR-T cells have shown limited success targeting solid tumors. One major hurdle is the immunosuppressive tumor microenvironment of solid tumors that promotes T-cell exhaustion. Another challenge is a poor understanding of how CAR structure impacts the signaling mechanisms driving T cell function, which has prevented the rational design of a superior CAR therapeutic with potent anti-tumor activity and resistance to exhaustion. I am a Damon Runyon Cancer Research Postdoctoral fellow at UCSF. My mentor is Dr. James Wells, an expert in chemical biology and protein engineering, and my co-mentor is Dr. Arthur Weiss, an expert in T cell biology. Tyrosine phosphorylation is the central component of the signaling pathways of CAR-T cells but the tools available to study pY modifications are very limited. During my postdoctoral training, I developed an innovative and generalizable platform called “pY-Targeting by Recombinant Antibody Pairs” or “pY-TRAP” to engineer highly specific and tight pY binding domains. This revolutionary tool is the first in vitro method for engineering specific binders against pY-modifications in three-dimensional protein structures. It provides a platform for developing strategies to engineer and to rewire pY-mediated signaling pathways in CAR-T cells. In this work, I propose to develop pY-TRAP-based strategies to address key challenges in CAR-T cell therapies. In Aim 1, I will determine how differing CAR structure modulates the CAR interactome using a novel pY-dependent proximity ligation approach. This work will reveal key differences in the CAR signalosome for various CAR designs and expose the mechanisms behind their functional divergence. In Aim 2, I will establish a screening method to determine how varying CAR structures affect the cellular response to PD-1 signaling. This work will further unveil how to rationally engineer CAR-T cells with enhanced resistance to immunosuppression. In Aim 3, I will engineer a synthetic PD-1 pathway in CAR-T cells designed to counteract the original immunosuppressive function of the pathway1. This is a fundamentally different approach relative to existing strategies to make CAR-T cells more resistant to TME-associated immunosuppressive signals. Taken together, the studies outlined in this proposal will lead to a deeper understanding of CAR-T cell biology, provide knowledge to inform future CAR designs, and expose new strategies to engineer and optimize signaling outcomes in cells to advance the development of cell therapeutics. With the support of my mentors, collaborators, consultants and the great research environment at UCSF, I will receive training in proteomics, quantitative data analysis, and T cell biology. These skills will help me reach my long-term goal of becoming the head of a laboratory performing rigorous scientific research investigating novel strategies to engineer the human immune system. 1 Aim 3 contains proprietary/privileged information that Dr. Xin Zhou requests not be released to persons outside the government, except for purposes of review and evaluation.