High throughput antibody discovery against cell membrane bound target proteins using innovative MOD technology for direct screening in single-cell assays

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&gt；100k 配对细胞分析在&lt；3小时内，与验证的概念。使用这项创新技术，SBIR阶段 1个项目提出了用于单细胞功能的测试方法的开发和量化 筛选工作流程，以支持大规模筛选治疗性抗体(Ab)候选者。最新进展 使用这样的工作流来可靠、一致且可重复地识别大量不同的B细胞命中池将 与经典但效率低下的杂交瘤方法相比，提供了显著的优势。将直接B细胞检测移植到 微流体是一种天然的选择，因为短命的B细胞可以迅速产生大量分泌的抗体 在适当小的体积中孵化时的浓度；目前的尝试受到成本和 可扩展性，并且没有一个提供针对目标细胞的高通量(HT)分析、敏感分析或集成的HT 测序。MOD代表了建立基于液滴的细胞分析的能力的进化进步 具有精确度和规模，有效地将分析构建、读数、命中选择和样品准备集成到 单一的工作流程和工具。MOD在同一微流控系统中共包裹抗体分泌细胞和靶细胞 水滴，这使得能够建立基于目标细胞的分析，因为它将携带抗体分泌细胞 因此，在随后的测序步骤中，可用于识别抗体的RNA。MOD利用流 细胞仪风格的检测和分类，因此它很容易扩展到HT。这个项目的方法一直是 从之前在MOD平台上开发测试的工作中获得了信息。在第一个目标中，将开发两种分析方法 检测抗体与膜蛋白靶标的结合。第一个将采用现有的基于珠子的免清洗 用于高拷贝数靶标的检测方案，第二种将开发一种更敏感的检测方法 低拷贝数目标与清洗步骤，并将探索适当的方法来创建持久的物理 细胞之间的连接将通过FACS分选或重新封装而持续。第二个目标将测试和 用免疫小鼠的B细胞对系统进行量化，以实现抗体发现的真实演示。B细胞将 来自以SARS-CoV-2为抗原的3组小鼠的标准四周免疫方案， 并将用于探索液滴中B细胞原代培养的参数和其他相关因素 将B细胞生物学移植到MOD平台上。将测试一小批(50-100K)B细胞/靶细胞分析， 假设平台的HT将与高命中率(~1000个阳性检测)相关，则 将通过生物信息选择少量(~10)候选抗体用于后续的重新克隆和点击 验证。成功的MOD抗体筛查将在以下能力方面引入新的范式 研究人员绕过目前成本的限制，确定更大和更多样化的抗体候选领域， 可伸缩性、商业可用性或技术复杂性，最终导致更好的治疗方法。

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 >100k paired-cell assays in <3 hours, with proven proof of concept. Using this innovative technology, this SBIR Phase 1 project proposes the development and quantification of assay methods to be used for single-cell functional screening workflows to enable large scale screening of therapeutic antibody (Ab) candidates. The development of such a workflow to reliably, consistently, and repeatably identify large and diverse pools of B cell hits would offer a significant advantage over the classical but inefficient hybridoma method. Porting direct B-cell assays to microfluidics is a natural fit because short lived B-cells can rapidly generate significant secreted Ab concentrations when incubated in appropriately small volumes; current attempts are limited by cost and scalability, and none offer high throughput (HT) assays against target cells, sensitive assays, or integrated HT sequencing. 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 Ab-secreting and target cells in the same microfluidic droplet, which enables building an assay based on the target cell, since it will carry along the Ab-secreting cell and therefore the RNA that is available to identify the Ab in a subsequent sequencing step. MOD utilizes flow cytometry-style detection and sorting, so it is readily scalable for HT. The approach for this project has been informed by previous work developing assays on the MOD platform. In the first aim, two assays will be developed to detect Ab binding against membrane protein targets. The first will adopt an existing bead-based no wash assay scheme for use with high copy number targets, and the second will develop a more sensitive assay for low copy number targets with a wash step, and will explore the appropriate method for creating a durable physical linkage between the cells that will last through FACS sorting or re-encapsulation. The second aim will test and quantify the system with B-cells from immunized mice for a real-world demonstration of Ab discovery. B-cells will be sourced from standard 4-week immunization protocols on groups of 3 mice using SARS-CoV-2 as the antigen, and will be used to explore the parameters of primary B-cell culture in droplets and other factors associated with porting B-cell biology on to the MOD platform. A small batch (50-100k) of B-cell/target cell assays will be tested, and assuming that the HT of the platform will correlate with a high number of hits (~1000 positive assays), a small number (~10) of Ab candidates will be bioinformatically selected for subsequent re-cloning and hit validation. Successful MOD-enabled antibody screening would introduce a new paradigm in the capabilities of researchers to identify a larger and more diverse field of Ab candidates, bypassing current limitations of cost, scalability, commercial availability, or technical complexity, and ultimately leading to better therapies.