Next-generation Ligand-dependent Transcriptional Switch Receptors for the Control and Customization of Cell Therapeutics

摘要 细胞因子免疫疗法和嵌合抗原受体-T细胞(CAR-ts)具有巨大的治疗潜力 诱导患者自身的免疫系统清除恶性病变。不幸的是，这些潜力中的许多 免疫治疗因疗效不佳和不可接受的毒性而受阻，如“靶点内、肿瘤外” 效应“，健康组织通过低水平表达肿瘤抗原而成为意外的CAR-T靶点。 与结构性CAR表达不同，诱导T细胞仅在 第二种肿瘤抗原的存在增加了对细胞毒反应的额外调节。最近 开发合成Notch(SynNotch)受体电路允许免疫治疗性表达 仅限于肿瘤部位。然而，这些受体不提供对表达的量的控制。 免疫治疗，也不允许调节产物的半衰期。能够调整音量和音量 诱导细胞免疫治疗的持续时间将进一步提高细胞免疫治疗的安全性 并为免疫治疗方案的稳健调整提供了一个平台。 到目前为止，在我的研究生培训中，几种新的受体设计产生了不同的水平和半衰期 已经设计了对表面表达的抗原做出反应的转录输出。通过受体 结构域调制，这些受体支架可以促使将设定数量的输出传递到设定的组织 在设定的时间量内键入。此外，某些新型受体起着“或”门的作用，可被任一种受体激活 配体或T细胞激活。对于这项提议，这些受体将被测试它们诱导表达的能力 各种潜在的免疫疗法，包括人类原始T细胞中的CARS和细胞因子的测量 采用酶联免疫吸附试验和流式细胞仪检测。此外，受体将作为受体-CAR正反馈的一部分进行测试 激活CAR-T细胞的回路，通过诱导更持久的 在细胞培养和小鼠模型中均有抗肿瘤反应。 加州大学旧金山分校(UCSF)是合成免疫学的领先机构， 它的教职员工在内科-科学家培训方面经验丰富。作为加州大学旧金山分校罗伊巴尔实验室的一名医学博士/博士生， 我可以获得世界知名研究人员的支持和合作，以及来自 帕克癌症免疫治疗研究所和陈·扎克伯格生物中心。除了我的博士研究， 我的培训计划包括在研究生培训期间与世界级医生一起担任医学导师， 通过加州大学旧金山分校医学院提供更多的教学机会，并通过 加州大学旧金山分校的资源，包括加州大学旧金山分校的布里奇斯医学课程。总体而言，这项培训提案力求进一步 开发合成受体回路作为提高潜在免疫疗法安全性和有效性的工具 同时为我提供了一系列不同的经验，为我未来的内科科学家职业生涯做准备。

ABSTRACT Cytokine immunotherapeutics and chimeric antigen receptor-T cells (CAR-Ts) carry great potential for inducing a patient’s own immune system to clear a malignant lesion. Unfortunately, many of these potential immunotherapies are hindered by poor efficacies and unacceptable toxicities, such as “on-target, off-tumor effects”, where healthy tissues become unintended CAR-T targets by expressing tumor antigens at low levels. In contrast to constitutive CAR expression, inducing a T cell to only express an immunotherapeutic in the presence of a second tumor antigen adds additional regulation over the cytotoxic response. A recent development, synthetic Notch (SynNotch) receptor circuits allow for immunotherapeutic expression to be restricted to tumor sites. However, these receptors do not provide control over the amount of expressed immunotherapeutic, nor do they allow for regulation of an output’s half-life. The ability to tune the level and duration of an induced cell-based immunotherapeutic would further improve on the safety of cell-based immunotherapies and provide a platform for robust tuning of an immunotherapeutic regimen. To date in my graduate training, several novel receptor designs that produce distinct levels and half-lives of transcriptional output in response to a surface-expressed antigen have been engineered. Through receptor domain modulation, these receptor scaffolds can prompt the delivery of a set amount of output to a set tissue type, for a set amount of time. In addition, certain novel receptors act as “OR” gates, being activatable by either ligand or T-cell activation. For this proposal, these receptors will be tested for their ability to induce the expression of various potential immunotherapeutics, including CARs and cytokines in human primary T-cells, as measured by ELISA and flow cytometry. In addition, receptors will be tested as part of receptor-CAR positive feedback circuits for their ability to prime CAR-T cells to better clear a heterogenous tumor by inducing a more sustained anti-tumor response, both in cell culture and in mouse model. The University of California, San Francisco (UCSF) is a leading institution in synthetic immunology, and its faculty are well experienced in physician-scientist training. As a MD/PhD student in the Roybal lab at UCSF, I have access to support and collaboration from world-renowned investigators, as well as resources from the Parker Institute for Cancer Immunotherapy and the Chan Zuckerberg BioHub. In addition to my PhD research, my training plan includes medical preceptorships with world-class physicians during my graduate training, additional teaching opportunities through the UCSF School of Medicine, and further clinical training through UCSF resources, including the UCSF Bridges medical curriculum. Overall, this training proposal seeks to further develop synthetic receptor circuits as a tool for improving the safety and efficacy of potential immunotherapies while providing me a diverse set of experiences in preparation for a future career as a physician-scientist.