Hydrogel microparticle technology for high-throughout screening of chimeric antigen receptor-T cells based on single cell effector function

摘要 与分子和基因干预一样，工程细胞疗法正在成为医学的支柱。在……里面 特别是，嵌合抗原受体(CAR)-T细胞疗法在过去的5年中获得了FDA的多项批准 多年来一直以血液系统恶性肿瘤为目标。延长这些疗法的成功，特别是对治疗 对于实体瘤，更彻底地了解CAR结构与CAR-T细胞功能是如何联系的 这是必要的。值得注意的是，从大量人群中分析和选择单个CAR-T细胞的广泛工具基于 严重缺乏对功能特性的影响，如分泌产物或细胞溶解活性。单细胞功能 检测可以筛选引入细胞池的汽车设计库，以识别罕见的功能 并对这些细胞进行分类，以恢复与重要效应器功能相关的汽车设计。如果在 高吞吐量，数千个构造可以通过每个构造的数百个单独事件进行筛选，以 在功能和序列之间的联系中具有强大的统计准确性。此外，如果单细胞功能 信息可以与转录信息捆绑在一起，途径分析与强大的效应功能相关 可以执行，识别其他改善功能的基因靶点，即使在存在 免疫抑制、易衰竭或其他与实体肿瘤相关的微环境。Partilion‘s 纳米瓶技术提供了一种新的方法来测量单个细胞的功能，使用广泛可用的 荧光激活细胞分类器(FAC)和单细胞测序仪，我们的目标是将其应用于细胞 治疗方面的发现。在这里，我们建议开发与单细胞分泌和细胞杀伤试验兼容的 纳米瓶公司将推出一种新产品，使数百万人的细胞治疗发现工作流程能够规模化 细胞。我们的目标是确定最佳的纳米瓶配方和程序，以测量细胞因子的产生和 来自相同细胞的细胞溶解功能，然后将单细胞转录信息与该功能联系起来 读数。我们还将设计纳米管，以更好地概括肿瘤微环境，充当人造的 结合抗原和免疫抑制信号的抗原提呈靶细胞。该方法 应该广泛适用于CAR-T细胞疗法以外的自然杀伤细胞中的其他嵌合受体，以及 巨噬细胞，或在寻找工程T细胞受体方面。最终，更多地获得复杂的细胞选择 治疗方法可以带来价格更低、效果更好以及扩大范围的疗法。 应用于未探索的治疗领域。

ABSTRACT Engineered cell therapies are becoming a pillar of medicine, along with molecular and genetic interventions. In particular, chimeric antigen receptor (CAR)-T cell therapies have had a number of FDA approvals in the last 5 years targeting hematologic malignancies. To extend the success of these therapies, especially for the treatment of solid tumors, a more thorough understanding of how CAR structure is linked to CAR-T cell function is necessary. Notably, widespread tools to analyze and select single CAR-T cells from a large population based on functional properties, such as secreted products or cytolytic activity, are critically lacking. Single-cell functional assays can enable screening a library of CAR designs introduced into a pool of cells to identify rare functional cells and sort these cells to recover CAR designs associated with important effector functions. If conducted in high throughput, thousands of constructs can be screened with hundreds of individual events per construct to have robust statistical accuracy in the linkage between function and sequence. In addition, if single-cell functional information can be tied to transcriptomic information, pathway analysis associated with strong effector functions can be performed, identifying other gene targets that improve function, even in the presence of immunosuppressive, exhaustion-prone, or other microenvironments associated with solid tumors. Partillion’s nanovial technology provides a new approach to measure the function of single cells using widely available fluorescence activated cell sorters (FACS) and single-cell sequencing instruments, which we aim to apply to cell therapy discovery. Here, we propose to develop single-cell secretion and cell-killing assays compatible with the nanovials to introduce a new product that would enable scaled cell therapy discovery workflows from millions of cells. We aim to identify optimal nanovial formulations and procedures to measure both cytokine production and cytolytic functions from the same cells, and then link single-cell transcriptomic information with this functional readout. We also will engineer nanovials to better recapitulate the tumor microenvironment, acting as an artificial antigen-presenting target cell with combinations of antigen and immunosuppressive signals. The approach should be applicable broadly beyond CAR-T cell therapies to other chimeric receptors in natural killer cells, and macrophages, or in finding engineered T cell receptors. Ultimately, more access to sophisticated cell selection approaches can lead to therapies that are both lower in price and more effective as well as expanding the scope of applications to un-explored therapeutic areas.