Engineering Protein Modulators of Notch Activation for T-cell immunotherapy

总结 明尼苏达大学的研究人员合作设计了Notch的新型蛋白质激活剂， 细胞表面受体家族，其是T细胞从诱导的多能分化的主要调节剂。 干细胞这项技术将加速用于治疗癌症的工程T细胞疗法的发展， 以及一系列疾病，包括自身免疫性疾病、传染病、免疫缺陷、移植物抗宿主病（graft vs. 宿主疾病和器官移植排斥现有FDA批准的工程T细胞功能强大 然而，主要的挑战仍然存在，包括难以将T细胞与前体细胞类型区分开来 以及在分化之前编辑和验证前体细胞的困难。为了克服这些局限性， 为了实现T细胞工程方法的变革性飞跃，研究小组正在开发试剂， 靶向并触发Notch 1的蛋白水解开关NRR结构域的构象打开。Notch NRR 掩埋了一个隐蔽的蛋白酶位点，该位点通常仅在生理上通过牵引力暴露于其蛋白酶 在其配体与邻近细胞结合期间产生。该项目旨在开发可溶性纳米抗体 功能性地撬开结构域并消除与Notch配体共培养的需要的试剂 在T细胞分化过程中。在该项目的目标1中，噬菌体展示蛋白工程用于鉴定和 优化选择性结合Notch 1 NRR的结构不同状态的纳米抗体。在Aim 2中， 纳米抗体在阵列中策略性地连接在一起，用于通过诱导 NRR的构象“开放”。在目标3中，报告Notch 1信号传导和T细胞定型的iPSC细胞系 将被开发、表征，然后用于监测iPSC分化为T细胞的测定， 通过工程化Notch 1激活剂诱导。在项目的最后阶段，报告细胞系将用于 比较目标1和目标2中开发的Notch 1激活剂的性能的基准测试实验， 行业标准技术。该项目的里程碑是：（a）生成一套分子工具， 选择性地结合Notch 1 NRR;（B）开发一组iPSC报告系统， 以及iPSC向具有正常生理功能（包括细胞杀伤潜力）的T细胞的分化;（c） 创造有效的和选择性的Notch 1激活剂，诱导iPSC分化为T细胞， 与目前的方法相比，效率差异和扩展方面的改进。这将使 开发更好的癌症治疗方法，并通过增加获得治疗的机会来改善健康差距 使用标准化的基于iPSC的细胞来源，可以冷冻并储存多个剂量， 降低每件产品的成本。

Summary Investigators at the University of Minnesota have teamed up to engineer novel protein activators of the Notch family of cell surface receptors, which are master regulators of T-cell differentiation from induced pluripotent stem cells. This technology will accelerate the development of engineered T cell therapies for treating cancer as well as a range of diseases including auto-immune disorders, infectious disease, immune-deficiencies, graft vs. host disease, and organ transplant rejection. Existing FDA approved engineered T cells are powerful therapeutics yet major challenges remain, including difficulty in differentiating T cells from precursor cell types and difficulty in editing and validating precursor cells prior to differentiation. To overcome these limitations and to enable a transformative jump in T cell engineering approaches, the research team is developing reagents that target and trigger conformational opening of the proteolytic switch NRR domain of Notch1. The Notch NRR buries a cryptic protease site that is normally only exposed to its protease physiologically by tugging forces generated during binding of its ligand on a neighboring cell. The project aims to develop soluble nanobody reagents that functionally pry open the domain and remove the requirement for co-culture with Notch ligands during T cell differentiation. In Aim 1 of the project, phage-display protein engineering is used to identify and optimize nanobodies that selectively bind structurally distinct states of the Notch1 NRR. In Aim 2, these nanobodies are strategically linked together in arrays for creating potent Notch1 activators by inducing conformational “opening” of the NRR. In Aim 3, iPSC cell lines that report Notch1 signaling and T cell commitment will be developed, characterized, and then used in assays monitoring differentiation of iPSCs into T cells under induction by engineered Notch1 activators. In the final stage of the project, the reporter cell lines will be used in benchmarking experiments comparing the performance of Notch1 activators developed in Aim 1 and 2, to industry standard technologies. The milestones of this project are: (a) generating a tool set of molecules that bind selectively to the Notch1 NRR; (b) developing a panel of iPSC reporter systems that monitor commitment and differentiation of iPSCs to T cells that have normal physiologic function including cell killing potential; (c) creating potent and selective Notch1 activators that induce iPSCs to differentiate into T cells with a marked improvement in the efficiency differentiation and expansion over current approaches. This will enable development of improved cancer treatments, and improve health disparities by increasing access to treatment with a standardized iPSC-based cell source that can be frozen and banked for multiple doses while significantly bringing down cost per product.