Engineering Machinery Molecules to Visualize and Actuate Immunotherapy

使免疫疗法可视化和重新编程的工程机械分子 摘要 过继免疫疗法有可能成为癌症治疗的范式转换技术。 具体地说，基于细胞的免疫疗法在针对各种疾病的临床试验中表现出了惊人的成功。 恶性肿瘤。尽管前景看好，但工程免疫细胞在靶向肿瘤方面的高度精确控制 在以细胞为基础的治疗能够被广泛采用之前，需要细胞。合成生物学是一个新兴的领域。 总体目标是使用工程方法来理解和操纵生命过程，尤其是 分子工程学。自然产生的分子和结构域可以被调制和整合来产生 具有良好控制功能的调节模块和分子机器。这将允许工程上的 能够检测抗原/生物标志物以激活细胞免疫的可控机械分子- 回应。基于荧光共振能量转移(FRET)的生物传感器已经彻底改变了 生物医学研究，允许对活细胞中的分子活动进行直接可视化和表征。这个 因此，机械分子及其模块化组件的官能度和功效可以 以FRET生物传感器为特征，作为“数字万用表”为用户提供即时反馈 高精尖机械分子的优化。我们的目标是设计集成的机械分子 它可以提供对细胞内空间的监视，可视化特定的 生物化学事件，并自动触发分子行动，指导免疫细胞的功能。我们有 采用模块化组装的方法开发了一种机械分子，专门用于传感 细胞内酪氨酸磷酸酶(PTP)Shp2的磷酸化和随后的激活，它扮演着一种 在各种病理生理过程中起关键作用。我们已经将这种机械分子进一步集成到 巨噬细胞中CD47受体Sirpα与Sirpα的结合及其激活 自然的负面信号将被重新连接，以打开积极的Shp2动作，以激活工程设计的 抗基因靶向抗体诱导巨噬细胞吞噬及其与Fcγ的相互作用 感受器。在这项提案中，我们计划应用这一策略来重新设计巨噬细胞，以根除 肿瘤。我们选择细胞高水平表达CD47的结肠癌作为我们的第一个概念验证目标。 因此，提出了两个具体目标： 具体目的1.鉴定重组细胞的吞噬效率及其相关的FRET信号 巨噬细胞抗结肠癌细胞。 特定目的2.检测重组巨噬细胞对裸鼠结肠癌的清除效果 和具有免疫能力的小鼠模型。 我们的平台设计为高度模块化，每个功能模块都可以随时切换以重新布线 分子网络。该平台允许新的肿瘤根除模式，具有可切换的抗体接口 原则上，任何类型的肿瘤都可以进行放射治疗。我们的工程化巨噬细胞还可以与传统的 放射治疗和化疗方法以及免疫治疗抗体方法可缓解 免疫抑制的PD-1和CTLA-4信号。调节基质细胞的维生素D类似物，以及透明质酸 酸性(HA)信号抑制剂PEGPH20促进细胞外基质(ECM)耗竭和肿瘤血管生成 可以调节肿瘤微环境。这些现有的方法也可以与我们的 工程化巨噬细胞方法提高肿瘤免疫治疗的治疗效率。因此， 拟议方法的成功将彻底改变进行基于细胞的免疫疗法的能力，并 突出在基础分子工程与临床医学之间架起桥梁的翻译力量。

Engineering Machinery Molecules to Visualize and Reprogram Immunotherapy Abstract Adoptive immunotherapy has the potential to become a paradigm shifting technology for cancer therapy. Specifically, cell based immunotherapy has demonstrated phenomenal success in clinical trials against various malignancies. Although promising, a high degree of precise control of engineered immunocells in targeting tumor cells is needed before the cell-based therapy can become widely adopted. Synthetic biology is an emerging field with the overall goal to understand and manipulate life processes using an engineering approach, particularly molecular engineering. Naturally occurring molecules and domains can be modulated and integrated to produce regulatory modules and molecular machines with well controlled functions. This will allow the engineering of controllable machinery molecules capable of detecting antigens/biomarkers to activate cellular immuno- responses. Biosensors based on fluorescence resonance energy transfer (FRET) have revolutionized the biomedical research by allowing direct visualization and characterization of molecular activities in live cells. The functionality and efficacy of the machinery molecules as well as their modular components can hence be precisely characterized by FRET biosensors serving as “digital multimeters” to provide immediate feedbacks for the optimization of the sophisticated machinery molecules. We aim to engineer integrated machinery molecules which can provide a surveillance of the intracellular space, visualizing the spatiotemporal patterns of specific biochemical events and automatically triggering molecular actions to guide immuno-cell functions. We have adopted a modular assembly approach to develop a machinery molecule, specifically for the sensing of intracellular phosphorylation and consequent activation of a tyrosine phosphatase (PTP) Shp2, which plays a critical role in various pathophysiological processes. We have further integrated this machinery molecule to the “don't eat me” CD47 receptor SIRPα in macrophages such that the engagement of SIRPα and its activation of naturally negative signals will be rewired to turn on a positive Shp2 action to activate the engineered macrophages and facilitate phagocytosis initiated by an anti-gen-targeting antibody and its interaction with Fcγ receptors. In this proposal, we plan to apply this strategy to re-engineer macrophages for the eradication of tumors. We choose colon cancer in which cells express a high level of CD47 as our first proof-of-concept target. Two specific aims are accordingly proposed: Specific Aim 1. Characterize the phagocytic efficiency and its associated FRET signals of re-engineered macrophages against colon cancer cells. Specific Aim 2. Examine the efficiency of re-engineered macrophages in eradicating the colon tumors in nude and immunocompetent mouse models. Our platform is designed to be highly modular, with each functional module readily switchable to rewire the molecular network. The platform allows new mode of tumor eradication, with a switchable antibody interface for the eradiation, in principle, any type of tumors. Our engineered macrophage can also be combined with traditional radiotherapy and chemotherapy approaches as well as the immunotherapy antibody methods mitigating the immuno-inhibitory PD-1 and CTLA-4 signals. Vitamin D analog in modulating stromal cells, and the hyaluronic acid (HA) signaling inhibitor PEGPH20 in promoting extracellular matrix (ECM) depletion and tumor vascularity can modulate tumor microenvironment. These existing methods can also be combined together with our engineered macrophage approach to improve the therapeutic efficiency of tumor immunotherapy. Therefore, the success of the proposed approach will revolutionize the ability to perform cell-based immunotherapy and highlight the translational power in bridging the fundamental molecular engineering to clinical medicine.