Microenvironment on Demand (MOD): A platform for single-cell cytotoxicity assays

按需微环境 (MOD)：单细胞细胞毒性测定平台

• 批准号: 10601155
• 负责人: Russell H Cole
• 金额: $ 35万
• 依托单位: SCRIBE BIOSCIENCES, INC.
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-07 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10601155
• 关键词: Academia; Benchmarking; Biological Assay; Biological Models; Biological Phenomena; Biological Sciences; Biology; CD19 gene; Categories; Cell Cycle Kinetics; Cell Line; Cell Therapy; Cell model; Cell secretion; Cells; Cellular Assay; Coculture Techniques; Comb animal structure; Cytotoxic T-Lymphocytes; Data; Detection; Development; Devices; Effector Cell; Encapsulated; Flow Cytometry; Genomics; Hour; Image; Immune; Immunology; Immunooncology; Immunotherapy; Incubated; Individual; Industry; Industry Standard; Interleukin-2; K-562; Kinetics; Knowledge; Label; Libraries; Malignant Neoplasms; Measurement; Measures; Mediating; Methods; Microfluidics; Modeling; Natural Killer Cells; Performance; Phase; Population; Preparation; Reagent; Research; Research Personnel; Sampling; Scheme; Small Business Innovation Research Grant; Sorting - Cell Movement; Stains; System; T cell therapy; Technology; Testing; Therapeutic; Time; Work; assay development; base; cell killing; cell type; chimeric antigen receptor; chimeric antigen receptor T cells; clinical development; combinatorial; cytotoxic; cytotoxicity; experimental study; fluorescence imaging; improved; in vivo; innovative technologies; instrument; microfluidic technology; phase 2 study; phenotypic data; screening; single cell sequencing; success

ABSTRACT Scribe Biosciences are leading experts in the field of droplet microfluidics and have developed a best-in-class droplet manipulation platform, Microenvironment on Demand (MOD), that can currently assemble more than 100k paired-cell assays in under 3 hours, with proven proof of concept. Using this innovative technology, this SBIR Phase 1 project proposes the development of a new functional screening platform that builds single-cell combinatorial assays to be used for workflows for cell therapy candidates, to be tested here with CAR T cells. The development of such a platform to reliably, consistently, and repeatably interrogate the activity of single CAR T cells would answer a significant research need: Currently, even though CAR T cell-based therapeutics is the largest category of immune-oncology agents under clinical development, there are considerable knowledge gaps regarding many key mechanisms governing their activity, compounded by limited industry-standard candidate discovery methods (bulk averages across an assay) which do not provide adequate information on important factors. MOD represents an evolutionary advancement in the capability to build droplet-based cell assays with precision and scale, effectively integrating assay construction, readouts, hit selection, and sample prep into a single workflow and instrument. MOD co-encapsulates effector and target cells in the same microfluidic droplet, easing identification of cytotoxic effector cells, and utilizes flow cytometry-style detection and sorting, so it is readily scalable for high throughput. The approach for this project has been informed by previous work developing assays on the MOD platform. Bulk interaction studies will be used to study cell killing kinetics and different assay reagents; results will be used to build robust cytotoxicity droplet-based screening assays for several model systems and quantify their performance. Natural killer (NK) cells and cytotoxic T-cells will be used, specifically NK92MI (IL-2 independent NK cell line) with K562 targets and anti-CD19 CAR-Ts with (CD19+) Nalm 6 targets. The second aim will seek to understand the sensitivity of the MOD assay workflow by benchmarking the droplet assay systems using spike-in experiments, using a cell system and assay reagent suitable for fast cell killing and slow killing assays (tested separately). The limits of the technology will be characterized as the relative amount of effector-cell containing droplets is incrementally reduced from 25% to1%. By building out assays for both fast and slow cell killing, the technology will be ready to be applied to many different interacting cell systems. Success of this functional screen assay will be determined by detection and sorting 10 hits of target cell killing by a 1% effector cell spike-in in both models, and will enable advancement to a Phase 2 study with diverse libraries and genomic integration. Successful MOD-enabled cytotoxicity assays could create a new paradigm in the type of specific data that can be extracted from interaction assays, and would significantly ease the identification of superior CAR T cell products with enhanced potency and persistence to improve efficacy and durability, and increase the breadth of treatable malignancies.

摘要 斯克里比生物科学公司是液滴微流体领域的领先专家，并已开发出一流的 液滴操纵平台，按需微环境(MOD)，目前可组装超过 100K配对细胞分析在3小时内完成，概念验证有效。使用这项创新技术，这 SBIR一期项目提出开发一种新的功能筛选平台，建立单细胞 用于细胞治疗候选者的工作流程的组合分析，这里将用CAR T细胞进行测试。 开发这样一种可靠、一致、可重复地询问单车活动的平台 T细胞将满足一个重要的研究需求：目前，尽管基于CAR T细胞的疗法是 处于临床开发中的最大类别的免疫肿瘤学药物，有相当大的知识差距 关于管理其活动的许多关键机制，再加上有限的行业标准候选人 发现方法(一次化验的总体平均值)不能提供足够的重要信息 各种因素。MOD代表了构建基于液滴的细胞分析的能力的进化进步 精密度和规模，有效地将分析构建、读数、命中选择和样品准备集成到一个 单一的工作流程和工具。MOD将效应器和靶细胞共同包裹在同一微流控液滴中， 简化了细胞毒性效应细胞的鉴定，并利用了流式细胞术式的检测和分类，因此 可随时扩展以实现高吞吐量。这个项目的方法已经从以前的工作中了解到了 在MOD平台上开发分析方法。整体相互作用研究将用于研究细胞杀伤动力学和 不同的检测试剂；结果将被用于建立基于液滴的强大的细胞毒性筛选分析 几个模型系统并量化它们的性能。将使用自然杀伤(NK)细胞和细胞毒性T细胞， 以K562为靶点的NK92MI(IL-2非依赖性NK细胞株)和(CD19+)NALM的抗CD19抗体 6个目标。第二个目标是通过基准测试来了解MOD检测工作流程的敏感性 液滴检测系统采用了尖峰实验，使用了适合FAST的细胞系统和检测试剂 细胞杀伤法和慢杀伤法(分别检测)。这项技术的局限性将被描述为 含有液滴的效应细胞的相对数量从25%逐渐减少到1%。通过扩建 对于快速和缓慢的细胞杀伤，这项技术将准备好应用于许多不同的相互作用 细胞系统。这种功能筛选试验的成功将取决于对10个靶点的检测和分类 在这两个模型中，通过1%的效应细胞刺激物杀死细胞，并将使研究进展到第二阶段 多样化的文库和基因组整合。成功的MOD激活的细胞毒性检测可能会创造出新的 可以从交互分析中提取的特定数据类型的范例，这将大大简化 具有增强效力和持久性以提高疗效的优秀CAR T细胞产品的鉴定 和耐用性，并增加可治疗的恶性肿瘤的广度。