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

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），目前可组装> 100 k 配对细胞分析在不到3小时内完成，经过验证的概念。利用这一创新技术， 1个项目提出了用于单细胞功能测定的测定方法的开发和定量， 筛选工作流程，以实现治疗性抗体（Ab）候选物的大规模筛选。发展 可靠地、一致地和可重复地鉴定大的和多样的B细胞命中池的这种工作流程将 提供了优于经典但低效的杂交瘤方法的显著优点。将直接B细胞检测移植到 微流体技术是一种天然的适合，因为短寿命的B细胞可以迅速产生大量的分泌性抗体， 当在适当小体积中孵育时，浓度是恒定的;目前的尝试受到成本和 可扩展性，并且没有一个提供针对靶细胞的高通量（HT）测定、灵敏测定或集成HT 测序MOD代表了建立基于液滴的细胞测定能力的进化进步 通过精确度和规模，有效地将检测构建、读数、命中选择和样品制备集成到 单一的工作流程和仪器。MOD将Ab分泌细胞和靶细胞共封装在同一微流体中 液滴，这使得能够建立基于靶细胞的测定，因为它将沿着Ab分泌细胞 因此在随后的测序步骤中可用于鉴定Ab的RNA。MOD利用流量 细胞计数式检测和分选，因此它很容易扩展用于HT。该项目的方法是 通过先前在MOD平台上开发分析的工作获得信息。在第一个目标中，将开发两种测定方法 以检测针对膜蛋白靶标的Ab结合。第一个将采用现有的珠为基础的不洗 第一个将开发用于高拷贝数靶标的检测方案，第二个将开发用于高拷贝数靶标的更灵敏的检测方案。 低拷贝数的目标与洗涤步骤，并将探索适当的方法，创造一个持久的物理 细胞之间的连接将持续通过FACS分选或重新封装。第二个目标将测试和 用来自免疫小鼠的B细胞对系统进行定量，以真实地展示Ab发现。B细胞将 来源于使用SARS-CoV-2作为抗原的3只小鼠的标准4周免疫方案， 并将用于探索液滴中原代B细胞培养的参数和与之相关的其他因素。 将B细胞生物学移植到MOD平台上。将检测一小批（50- 100 k）B细胞/靶细胞测定， 并假设平台的HT将与大量命中（~1000次阳性测定）相关， 将通过生物信息学方法选择少量（约10个）候选Ab用于后续的重新克隆和命中 验证。成功的MOD使能抗体筛选将在免疫功能方面引入新的范例。 研究人员可以确定更大和更多样化的抗体候选领域，绕过目前的成本限制， 可扩展性、商业可用性或技术复杂性，并最终导致更好的治疗。

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.