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

项目摘要/摘要 工程化治疗性T细胞在治疗B细胞癌方面取得了革命性的成功，但应用这一技术 事实证明，实体肿瘤的治疗要困难得多。似乎没有绝对的肿瘤特异性单一 针对实体癌的抗原靶点，因此攻击大多数肿瘤相关抗原的CAR T细胞导致了 与表达该抗原的正常器官发生毒性交叉反应。如果我们要成功和安全地治疗 对于带有CAR T细胞的实体肿瘤，缓解与正常组织的毒性交叉反应将是必不可少的。 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细胞电路，以 根据多抗原特征识别肿瘤。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超越和抑制CAR T细胞激活和杀伤的电路 这是正常组织发生交叉反应的唯一标志(即肿瘤中缺少抗原)。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以用作 在大脑和肺等常见的交叉反应组织中，诱导T细胞失活的信号。尽管如此，在那里 目前缺乏在肿瘤模型中被证明工作良好的健壮的非门电路。因此，我们将发展 并测试能够以抗原诱导的方式使CAR T细胞失活的新的NOT电路。我们的具体目标是： 目标1.设计、制作和优化新的非门电路，使用不同的机制来阻止 抗原诱导的治疗性T细胞活化 目标1.1。T细胞不使用CAR表达的转录抑制因子。 目标1.2。T细胞不是通过抗原诱导细胞死亡效应来抑制T细胞增殖的门。 目标1.3。T细胞不是局部诱导产生分泌性免疫抑制因子的门(旁分泌) 目的2.应用NOT门电路防止抗GD2 CAR T与脑/中枢组织的交叉反应。 目标2.1。靶向脑抗原MOG关闭抗GD2 Car的NOT门电路的体外原型研究 目标2.2。在体内检测脑非门是否阻断抗GD2 CAR T细胞的中枢毒性，同时维持 抗小鼠神经母细胞瘤异种移植模型的疗效(GD2+)。 目标2.3。Not Gate CAR T细胞在小鼠免疫活性模型中的体内安全性和有效性检测 神经母细胞瘤。 这项工作应该为工程汽车T细胞提供重要的通用能力，这些细胞选择性地 当他们在错误的、交叉反应的组织中时，他们自己就会脱掉。这些都是非常需要的工具 目前在T细胞工程工具箱中缺失，但对于安全地设计T细胞将是至关重要的 治疗实体癌症。

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.