Ameliorating off-target toxicities of CAR T cells by engineering NOT gates

项目总结/摘要 工程化的治疗性T细胞在治疗B细胞癌症方面已经显示出变革性的成功，但应用这种方法， 实体瘤的治疗方法被证明要困难得多。似乎没有绝对的肿瘤特异性单一 因此，攻击大多数肿瘤相关抗原的CAR T细胞已经导致 与同样表达抗原的正常器官的毒性交叉反应。如果我们要成功安全地治疗 对于具有CAR T细胞的实体瘤，减轻与正常组织的毒性交叉反应至关重要。 为了防止治疗性T细胞的肿瘤外毒性，我们建议设计多受体T细胞回路， 基于多抗原谱识别肿瘤。在本提案中，我们特别关注工程，而不是 门电路--在检测到抗原时可以覆盖和抑制CAR T细胞激活和杀伤的电路 其唯一地指示交叉反应的正常组织（即，肿瘤中不存在抗原）。我们最近 已发表的生物信息学分析表明，有许多组织特异性抗原可用作 信号诱导T细胞失活常见的交叉反应组织，如脑和肺。尽管如此， 目前缺乏在肿瘤模型中表现出良好工作的鲁棒的非门电路。因此，我们将开发 并测试可以以抗原诱导的方式激活CAR T细胞的新NOT电路。我们的具体目标是： 目标1.设计、制作原型并优化新的非门电路，这些电路使用不同的机制来阻止 以抗原诱导方式的治疗性T细胞活化 目标1.1。使用CAR表达的转录阻遏物的T细胞NOT门控。 目标1.2。通过抗原诱导细胞死亡效应物抑制T细胞增殖的T细胞非门。 目标1.3。局部诱导分泌的免疫抑制因子产生的T细胞非门（旁分泌） 目标二。应用非门电路以防止抗GD 2 CAR T与脑/CNS组织的交叉反应。 目标2.1。靶向脑抗原MOG以关闭抗GD 2 CAR的NOT门电路的体外原型设计 目标2.2。测试脑非门控是否在体内阻断抗GD 2 CAR T细胞的CNS毒性，同时维持 对鼠神经母细胞瘤异种移植模型（GD 2+）的功效。 目标2.3。测试非门控CAR T细胞在免疫活性模型中的体内安全性和功效。 神经母细胞瘤 这项工作应该为工程化CAR T细胞提供重要的通用能力， 当它们在错误的交叉反应组织中时，它们自己关闭。这些都是非常需要的工具， 目前在T细胞工程的工具箱中缺少，但对于安全地工程化T细胞至关重要。 治疗实体癌

Project Summary/Abstract Engineered therapeutic T cells have shown transformative success in treating B cell cancers but applying this approach to solid tumors has proven far more difficult. There do not appear to be absolutely tumor-specific single antigen targets for solid cancers, and thus, CAR T cells that attack most tumor-associated antigens have led to toxic cross-reaction with normal organs that also express the antigen. If we are to successfully and safely treat solid tumors with CAR T cells, it will be essential to mitigate toxic cross-reaction with normal tissues. To prevent off-tumor toxicity of therapeutic T cells, we propose to engineer multi-receptor T cell circuits that can recognize a tumor based on a multi-antigen profile. In this proposal, we focus specifically on engineering NOT gate circuits -- circuits that can override and inhibit CAR T cell activation and killing upon detecting an antigen that is uniquely indicative of a cross-reactive normal tissue (i.e., antigen is absent in the tumor). Our recently published bioinformatic analysis shows that there are numerous tissue-specific antigens that could be used as signals to induce T cell inactivation in common cross-reactive tissues like the brain and lung. Nonetheless, there is currently a lack of robust NOT-gate circuits demonstrated to work well in tumor models. Thus, we will develop and test new NOT circuits that can inactivate a CAR T cell in an antigen-induced manner. Our specific aims are: Aim 1. Engineer, prototype and optimize new NOT gate circuits that use diverse mechanisms to block therapeutic T cell activation in antigen-induced manner Aim 1.1. T cell NOT gates using transcriptional repressors of CAR expression. Aim 1.2. T cell NOT gates that inhibit T cell proliferation by antigen-induction of cell death effectors. Aim 1.3. T cell NOT gates that locally induce production of secreted immunosuppressive factors (paracrine) Aim 2. Applying NOT gate circuits to prevent anti-GD2 CAR T cross-reaction with brain/CNS tissue. Aim 2.1. in vitro prototyping of NOT gate circuit targeting the brain antigen MOG to turn off anti-GD2 CAR Aim 2.2. Test if brain NOT gates block CNS toxicity of anti-GD2 CAR T cells in vivo, while maintaining efficacy against murine neuroblastoma xenograft models (GD2+). Aim 2.3. Test in vivo safety & efficacy of NOT gate CAR T cells in an immunocompetent model of neuroblastoma. This work should provide important general capabilities for engineering CAR T cells that selectively turn themselves OFF when they are in the wrong, cross-reactive tissue. These are much needed tools that are currently missing in the toolbox for T cell engineering, but which will be critical for engineering T cells that safely treat solid cancers.