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

项目摘要 嵌合抗原受体（CAR）-T细胞疗法在患有B-淋巴瘤的患者中取得了前所未有的成功。 细胞恶性肿瘤，在某些情况下表现出超过100%的完全缓解率。几乎所有这些 然而，患者随后复发，具有由抗原逃逸引起的治疗抗性肿瘤。在 此外，CAR-T细胞疗法的广泛应用已经满足了额外的安全性和有效性 挑战，包括靶向/肿瘤外毒性，由过度活跃的CAR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细胞应答， 避开这些障碍，避开非癌性旁观者组织，限制危险的副作用， 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CAR-T细胞活性以调节CAR-T细胞应答， 这种平衡。 为了满足这些需求，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性细胞应答的PAGER （基于GPCR的可编程粘附，外部调节）。为了进一步满足这些需求，我还将 为现有的CAR增加药物可控性。为了创建PAGER，我将设计一个粘附G蛋白偶联 受体（GPCR），使得其在激活时的结构重排与融合的GPCR的活性偶联。 蛋白酶，允许其在受体激活时释放转录因子。此外，添加药物- 为了提高PAGER和现有CAR的可控性，我将添加一种可药物化的蛋白酶依赖性降解， 向合成受体发信号以产生"药物作用"可调的PAGER和CAR。我会利用高通量 筛选、蛋白质工程和定向进化来实现这些目标。 药物控制的PAGER和CAR在CAR-T细胞中的应用将允许对细胞内的细胞因子进行微调调节。 CAR-T细胞活性，以限制或防止不需要的CAR-T细胞应答。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， 前所未有的决心。PAGER将用于控制CAR表达和活性、细胞因子分泌 谱，以及以用户控制的细胞自主方式局部递送治疗性抗体。宽 可以使用PAGER编程的细胞反应谱突出了它在帮助 克服目前CAR-T细胞疗法面临的许多安全性和有效性挑战。

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.