Engineering Protein Modulators of Notch Activation for T-cell immunotherapy

用于 T 细胞免疫治疗的 Notch 激活的工程蛋白质调节剂

基本信息

批准号：
10612995
负责人：
WENDY RYAN GORDON
金额：
$ 38.48万
依托单位：
UNIVERSITY OF MINNESOTA
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-08-01 至 2026-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10612995
关键词：
Acceleration Affinity Allogenic Antibodies Autoimmune Diseases Back Bacteriophages Benchmarking Binding Binding Proteins Biochemical Biochemistry Biological Assay Blocking Antibodies Cations Cell Differentiation Induction Cell Line Cell Nucleus Cell Surface Receptors Cells Cellular biology Clinical Clinical Trials Closure by clamp Coculture Techniques Collecting Cell Communicable Diseases Cultured Cells Cyclic GMP Development Disease Disulfides Dose Engineering Exposure to Extracellular Domain FDA approved Family Freezing Generations Goals Health Services Accessibility Hematologic Neoplasms Homologous Gene Immune Immune System Diseases Immunoglobulin Fragments Immunotherapeutic agent Immunotherapy Industry Standard Ligand Binding Ligands Link Malignant Neoplasms Measures Minnesota Molecular Conformation Monitor Mutation NOTCH3 gene Oncology Patients Peptide Hydrolases Performance Phage Display Physiological Population Protein Engineering Proteins Proteolysis Reagent Reporter Reporting Research Research Personnel Signal Transduction Site Source Standardization Structure Surface System T cell differentiation T cell therapy T-Cell Activation T-Lymphocyte Technology Testing Therapeutic Time Transcription Coactivator Transcription Initiation Universities Yeasts arm cancer cell cancer therapy cell killing cell type chelation chimeric antigen receptor chimeric antigen receptor T cells cost cost effective design engineered T cells experimental study graft vs host disease health disparity improved individual patient induced pluripotent stem cell innovation manufacturing process mimetics nanobodies next generation notch protein novel organ transplant rejection precursor cell programs protein phosphatase inhibitor-2 receptor structural biology surface coating therapeutic development tool

项目摘要

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.

概括明尼苏达大学的研究人员联手设计了新型 Notch 蛋白激活剂细胞表面受体家族，是诱导多能性 T 细胞分化的主要调节因子该技术将加速用于治疗癌症的工程化 T 细胞疗法的开发。以及一系列疾病，包括自身免疫性疾病、传染病、免疫缺陷、移植物抗病毒等。现有 FDA 批准的工程 T 细胞功能强大。治疗方法尚未成熟，但仍存在重大挑战，包括难以区分 T 细胞与前体细胞类型以及在分化前编辑和验证前体细胞的困难，以克服这些限制和为了实现 T 细胞工程方法的变革性飞跃，研究团队正在开发能够目标并触发 Notch1 蛋白水解开关 NRR 结构域的构象打开。埋藏了一个隐秘的蛋白酶位点，该位点通常仅通过拉力在生理上暴露于其蛋白酶该项目旨在开发可溶性纳米抗体。功能性撬开结构域并消除与 Notch 配体共培养的要求的试剂在该项目的目标 1 中，噬菌体展示蛋白工程用于识别和在目标 2 中，优化选择性结合 Notch1 NRR 的结构不同状态的纳米抗体。纳米抗体在阵列中战略性地连接在一起，通过诱导产生有效的 Notch1 激活剂 NRR 的构象“开放” 在目标 3 中，报告 Notch1 信号传导和 T 细胞定向的 iPSC 细胞系。将被开发、表征，然后用于监测 iPSC 分化为 T 细胞的检测在该项目的最后阶段，报告细胞系将用于通过工程化的 Notch1 激活剂进行诱导。基准测试实验比较了目标 1 和 2 中开发的 Notch1 激活剂的性能，以该项目的里程碑是：(a) 生成一套分子工具，选择性结合 Notch1 NRR；(b) 开发一组监控承诺的 iPSC 报告系统 iPSC 分化为具有正常生理功能（包括细胞杀伤潜力）的 T 细胞（c）创造有效且选择性的 Notch1 激活剂，诱导 iPSC 分化为具有标记的 T 细胞这将使现有方法的效率差异化和扩展得到改善。开发改进的癌症治疗方法，并通过增加获得治疗的机会来改善健康差距具有标准化的基于 iPSC 的细胞源，可以冷冻并储存多次剂量，同时显着降低每件产品的成本。