Bioengineering programmable and drug-controllable synthetic receptors fortunable CAR-T cell behaviors

项目摘要 嵌合抗原受体(CAR)-T细胞疗法在B细胞白血病患者中取得了前所未有的成功。 细胞恶性肿瘤，部分病例完全缓解率达100%以上。几乎所有这些 然而，患者随后会复发，出现由抗原逃逸引起的耐药肿瘤。在……里面 此外，CAR-T细胞疗法的广泛应用也带来了额外的安全性和有效性 挑战，包括靶上/肿瘤外毒性，由过度活跃的汽车引起的细胞因子释放综合征- T细胞和T细胞耗竭。需要精确、平衡和受控的CAR-T细胞反应 克服这些障碍，避免非癌症的旁观者组织，限制危险的副作用，并仍然 识别并摧毁肿瘤细胞。新的治疗方法，允许用户控制，微调 需要调节细胞自主的CAR-T细胞活性来调节CAR-T细胞的反应以维持 这种平衡。 为了满足这些需求，并通过本提案中概述的实验，我将创建一个系统 用于可编程和药物可控的抗原依赖细胞反应的寻呼机 (基于可编程黏附GPCR，外源调控)。为了进一步满足这些需求，我还将 增加现有汽车的药物可控性。为了创建寻呼机，我将设计一种粘连G蛋白偶联的 受体(GPCR)，使其激活时的结构重排与融合蛋白的活性偶联 蛋白水解酶，允许它在受体激活时释放转录因子。此外，要增加药物- 对寻呼机和现有汽车的可控性，我将添加一种依赖于蛋白酶的可药物降解 向合成感受器发出信号，以创造可调谐的“毒品”寻呼机和汽车。我将利用高吞吐量 筛选、蛋白质工程和定向进化来实现这些目标。 在CAR-T细胞中应用药物控制寻呼机和CARS将允许微调调节 CAR-T细胞活性，以限制或防止不需要的CAR-T细胞反应。我将演示概念验证 使用这些合成受体精确控制定制的CAR-T细胞行为 史无前例的分辨率。寻呼机将用于控制CAR的表达和活性，细胞因子的分泌 以及以用户控制的细胞自主方式局部递送治疗性抗体。宽阔的 可以使用寻呼机编程的细胞反应光谱突出了它的巨大潜力 在克服目前面临的CAR-T细胞治疗的许多安全性和有效性挑战方面。

Project Summary Chimeric Antigen Receptor (CAR)-T cell therapy has had unprecedented success in patients with B- cell malignancies, demonstrating upwards of 100% complete remission rate in some cases. Nearly all these patients, however, subsequently relapse with therapy-resistant tumors caused by antigen escape. In addition, the widespread utility of CAR-T cell therapy has been met with additional safety and efficacy challenges, including on-target/off-tumor toxicity, cytokine release syndrome caused from hyperactive CAR- T cells, and T cell exhaustion. A precise, well-balanced, and controlled CAR-T cell response is required to navigate these obstacles, avoid non-cancerous bystander tissues, limit dangerous side effects, and still identify and destroy tumor cells. New therapeutic approaches that permit the user-controlled, fine-tuned regulation of cell-autonomous CAR-T cell activity are needed to tune the CAR-T cell response to maintain this balance. To address these needs, and through the experiments outlined in this proposal, I will create a system for programmable and drug-controllable antigen-dependent cellular response coined PAGER (Programmable Adhesion GPCR-based, Exogenously Regulated). To further meet these needs, I will also add drug controllability to existing CARs. To create PAGER, I will engineer an adhesion G Protein-Coupled Receptor (GPCR) so that its structural rearrangements upon activation are coupled to the activity of a fused protease, allowing it to release a transcription factor upon receptor activation. Furthermore, to add drug- controllability to both PAGER and existing CARs, I will add a druggable protease-dependent degradation signal to synthetic receptors to create “drug-on” tunable PAGERs and CARs. I will utilize high-throughput screening, protein engineering, and directed evolution to accomplish these goals. Application of drug-controlled PAGERs and CARs in CAR-T cells will permit fine-tuned regulation of CAR-T cell activity to limit or prevent unwanted CAR-T cell responses. I will demonstrate proof-of-concept for using these synthetic receptors to precisely control customized CAR-T cell behaviors to an unprecedented resolution. PAGER will be used to control CAR expression and activity, cytokine secretion profile, and local delivery of therapeutic antibodies in a user-controlled cell-autonomous manner. The wide spectrum of cellular responses that can be programmed using PAGER highlights its great potential to assist in overcoming many of the safety and efficacy challenges that currently face CAR-T cell therapy.