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

具有合成磷酸酪氨酸识别域的 CAR-T 细胞中询问和重新布线细胞信号通路

基本信息

批准号：
10617843
负责人：
Xin Zhou
金额：
$ 24.9万
依托单位：
DANA-FARBER CANCER INST
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-15 至 2025-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10617843
关键词：
3-Dimensional Address Advanced Development Affect Affinity Antigens Attenuated Automobile Driving Binding Biology CAR T cell therapy Cell Line Cell physiology Cells Cellular biology Cellular immunotherapy Chemicals Complex Cytoplasm Data Analyses Development Engineering Environment Enzymes Evaluation Event Future Goals Government Head Hematologic Neoplasms Hematopoietic Neoplasms Human Engineering Immune system Immunosuppression In Situ In Vitro Individual Jurkat Cells Knowledge Label Laboratories Libraries Ligation Malignant Neoplasms Mediating Mentors Methods Modification Outcome Output PD-1 pathway Pathway interactions Persons Phase Phosphoric Monoester Hydrolases Phosphorylation Phosphotransferases Phosphotyrosine Postdoctoral Fellow Process Protein Engineering Proteins Proteomics Recombinant Antibody Recombinants Research Resistance Safety Signal Pathway Signal Transduction Signaling Protein Solid Neoplasm Specificity Structure T-Lymphocyte Technology Testing Therapeutic Training Tumor Promotion Tumor-associated macrophages Tyrosine Phosphorylation Work anti-cancer anticancer research cancer immunotherapy chimeric antigen receptor chimeric antigen receptor T cells cytokine design exhaustion improved innovation inorganic phosphate mutant novel novel strategies post-doctoral training prevent programmed cell death ligand 1 programmed cell death protein 1 programs protein structure rational design recruit response screening skills success synthetic biology targeted treatment tool translational approach tumor tumor microenvironment tumor-immune system interactions

项目摘要

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.

项目概要在治疗造血系统恶性肿瘤的最新突破中，嵌合抗原受体（CAR）-T 细胞疗法是癌症免疫疗法中最有前途的进展之一，然而，CAR-T 细胞具有以下优势。针对实体瘤的成功有限，其中一个主要障碍是免疫抑制肿瘤。促进 T 细胞耗竭的实体瘤微环境的另一个挑战是了解不足。 CAR 结构如何影响驱动 T 细胞功能的信号机制，从而阻止了合理设计具有有效抗肿瘤活性和抗衰竭性的优质 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。化学生物学和蛋白质工程专家，我的共同导师是T细胞专家Arthur Weiss博士生物学上，酪氨酸磷酸化是 CAR-T 细胞信号通路的核心组成部分。在我的博士后培训期间，可用于研究 pY 修饰的工具非常有限。称为“pY-Targeting by Recombinant Antibody Pairs”或“pY-TRAP”的创新和通用平台这种革命性的工具是设计高度特异性和紧密的 pY 结合域的第一个体外方法。针对三维蛋白质结构中的 pY 修饰设计特异性结合剂。用于开发策略来设计和重新连接 CAR-T 细胞中 pY 介导的信号通路的平台。在这项工作中，我建议开发基于 pY-TRAP 的策略来解决 CAR-T 细胞的关键挑战在目标 1 中，我将使用一种新颖的方法确定不同的 CAR 结构如何调节 CAR 相互作用组。 pY 依赖性邻近连接方法将揭示 CAR 信号体的关键差异。在目标 2 中，我将建立各种 CAR 设计并揭示其功能差异背后的机制。一种确定不同 CAR 结构如何影响细胞对 PD-1 信号传导反应的筛选方法。这项工作将进一步揭示如何合理设计具有增强抗性的 CAR-T 细胞。在目标 3 中，我将在 CAR-T 细胞中设计一条合成的 PD-1 通路，旨在抵消免疫抑制。通路的原始免疫抑制功能1 这是一种根本不同的方法。现有策略使 CAR-T 细胞对 TME 相关的免疫抑制信号具有更强的抵抗力。总之，该提案中概述的研究将导致对 CAR-T 细胞生物学的更深入了解，提供知识来指导未来的 CAR 设计，并公开新的策略来设计和优化细胞中的信号转导结果可促进细胞疗法的发展。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目标。我的长期目标是成为实验室负责人，进行严格的科学研究改造人类免疫系统的新策略。 1 目标 3 包含周鑫博士要求不得向政府以外人员透露的专有/特权信息，除非出于以下目的：审查和评价。