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 Scribe Biosciences是液滴微流体领域的领先专家，并已开发出一流的 液滴操作平台，按需微环境（MOD），目前可以组装超过 在3小时内完成100 k配对细胞分析，并经过验证的概念。利用这项创新技术， SBIR第1阶段项目提出开发一种新的功能筛选平台， 组合测定用于细胞疗法候选物的工作流程，在此用CAR T细胞进行测试。 开发这样一个平台，以可靠、一致和可重复地询问单个CAR的活性， T细胞将满足一个重要的研究需求：目前，即使基于CAR T细胞的治疗方法是最好的方法， 作为临床开发中最大的一类免疫肿瘤药物， 关于管理其活动的许多关键机制，加上有限的行业标准候选人， 发现方法（整个检测试剂盒的整体平均值）不能提供关于重要 因素MOD代表了建立基于液滴的细胞测定的能力的进化进步， 精确度和规模，有效地将检测构建、读数、命中选择和样品制备集成到一个 单一工作流程和仪器。MOD将效应细胞和靶细胞共包封在同一微流体液滴中， 易于细胞毒性效应细胞的鉴定，并利用流式细胞术式检测和分选，因此， 易于扩展以获得高吞吐量。该项目的方法已被告知以前的工作 在MOD平台上开发分析。本体相互作用研究将用于研究细胞杀伤动力学， 不同的测定试剂;结果将用于建立稳健的基于细胞毒性液滴的筛选测定， 几个模型系统，并量化其性能。将使用自然杀伤（NK）细胞和细胞毒性T细胞， 特别是具有K562靶标的NK 92 MI（IL-2非依赖性NK细胞系）和具有（CD 19+）Nalm的抗CD 19 CAR-T 6个目标。第二个目标是通过基准测试了解MOD检测工作流程的灵敏度 液滴测定系统使用掺加实验，使用适于快速测定的细胞系统和测定试剂， 细胞杀伤和缓慢杀伤测定（分别测试）。该技术的局限性将被描述为 含有效应细胞的液滴的相对量从25%逐渐减少到1%。通过构建 快速和缓慢的细胞杀伤试验，该技术将准备应用于许多不同的相互作用， 细胞系统该功能筛选试验的成功将通过检测和分选10个靶点来确定 在两种模型中，通过1%效应细胞加标实现细胞杀伤，并将能够推进到II期研究， 多样的文库和基因组整合。成功的MOD激活的细胞毒性试验可以创建一个新的 这是可以从相互作用测定中提取的特定数据类型的范例，并且将显著地简化 鉴定具有增强的效力和持久性以改善功效的上级CAR T细胞产物 和持久性，并增加可治疗恶性肿瘤的范围。