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

• 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
• 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

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.

项目摘要 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 显示出有限的针对实体瘤的成功。一个主要的障碍是免疫抑制肿瘤 实体肿瘤的微环境，促进T细胞耗竭。另一个挑战是缺乏理解 CAR结构如何影响驱动T细胞功能的信号传导机制，这阻止了T细胞的功能。 合理设计具有有效抗肿瘤活性和抗耗竭性的上级CAR治疗剂。 我是加州大学旧金山分校的达蒙·鲁尼恩癌症研究博士后。我的导师是詹姆斯威尔斯博士， 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细胞专家亚瑟韦斯博士 生物学酪氨酸磷酸化是CAR-T细胞信号传导途径的中心组分，但酪氨酸磷酸化是CAR-T细胞信号传导途径的核心组分。 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，我开发了一种 称为“重组抗体对的pY靶向”或“pY-TRAP”的创新和可推广的平台， 设计高度特异性和紧密的pY结合结构域。这种革命性的工具是第一种体外方法， 工程化针对三维蛋白质结构中的pY修饰的特异性结合剂。它提供了一个 该平台用于开发在CAR-T细胞中工程化和重新连接pY介导的信号通路的策略。 在这项工作中，我建议开发基于pY-TRAP的策略来解决CAR-T细胞中的关键挑战。 治疗在目标1中，我将确定不同的CAR结构如何使用一种新的方法调节CAR相互作用组。 pY依赖性邻近连接方法。这项工作将揭示CAR信号体的关键差异， 各种CAR设计，并揭示其功能差异背后的机制。在目标2中，我将建立 一种筛选方法，以确定不同的CAR结构如何影响对PD-1信号传导的细胞应答。 这项工作将进一步揭示如何合理地工程化CAR-T细胞，使其具有增强的抵抗力， 免疫抑制在目标3中，我将在CAR-T细胞中设计一种合成的PD-1通路， pathway 1的原始免疫抑制功能。这是一个根本不同的方法相对于 现有的策略使CAR-T细胞对TME相关的免疫抑制信号更具抗性。采取 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理解， 为未来的CAR设计提供信息，并为工程师和优化提供新的策略 细胞中的信号传导结果，以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 我的长期目标是成为一个实验室的负责人，进行严格的科学研究， 设计人类免疫系统的新策略。 1目标3包含专有/特许信息，周欣博士要求不得向政府以外的人发布，除非出于以下目的 审查和评价。