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

使用创新的 MOD 技术发现针对细胞膜结合靶蛋白的高通量抗体，用于单细胞测定中的直接筛选

• 批准号: 10698891
• 负责人: Russell H Cole
• 金额: $ 30万
• 依托单位: SCRIBE BIOSCIENCES, INC.
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10698891
• 关键词: 2019-nCoV; Adopted; Antibodies; Antibody Therapy; Antigen-Presenting Cells; Antigens; B-Lymphocytes; Binding; Bioinformatics; Biological Assay; Biological Sciences; Biology; Biotin; Bypass; Cardiology; Cell Culture Techniques; Cell Line; Cell membrane; Cell secretion; Cells; Cellular Assay; Cellular biology; Chinese Hamster Ovary Cell; Cloning; Computer Analysis; Detection; Development; Devices; Disparate; Encapsulated; Engineering; Ensure; Flow Cytometry; G-Protein-Coupled Receptors; Genomics; High-Throughput Nucleotide Sequencing; Hour; Hybridomas; Hydrogels; Immunization; Immunize; Immunoglobulin-Secreting Cells; Inbred BALB C Mice; Incubated; Infection; Jurkat Cells; Malignant Neoplasms; Medical; Membrane Proteins; Methods; Microfluidics; Molecular Biology; Monoclonal Antibodies; Mus; Neurology; Oligonucleotides; Performance; Permeability; Phase; Population; Preparation; Process; Property; Proteins; Protocols documentation; RNA; Reagent; Research; Research Personnel; Running; SARS-CoV-2 spike protein; Sampling; Scheme; Series; Small Business Innovation Research Grant; Sorting; Source; Speed; Stains; Surface; Suspensions; System; T-Lymphocyte; Techniques; Technology; Testing; Therapeutic; Therapeutic antibodies; Titrations; Validation; Variant; Work; antibody and antigen binding; antibody detection; antibody test; assay development; combinatorial; commercialization; cost; experimental study; flexibility; high throughput screening; innovation; innovative technologies; instrument; lentivirally transduced; microfluidic technology; new technology; screening; single-cell RNA 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 >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.

抽象的 Scribe Biosciences是液滴微流体领域的主要专家，并且已经开发了一流的 液滴操纵平台，按需微环境（MOD），目前可以组装> 100K 在<3小时内配对细胞测定法，并具有可靠的概念证明。使用这种创新技术，这个SBIR阶段 1项目提出了用于单细胞功能的测定方法的开发和量化 筛选工作流程以实现治疗抗体（AB）候选的大规模筛查。发展 在这样的工作流程中，可以可靠，一致且重复地识别大量和多样化的B细胞命中池 比经典但效率低下的杂交瘤方法具有显着优势。将直接B细胞测定移植到 微流体是一种自然的拟合 浓度在适当的小体积中孵育；当前尝试受费用的限制， 可伸缩性，没有任何针对目标细胞，敏感测定或集成的HT的高通量（HT）测定 测序。 MOD代表了构建基于液滴的单元测定能力的进化进步 具有精度和规模，有效地整合了测定构建，读数，命中选择和样本准备 单个工作流程和仪器。 mod共同封闭AB分泌和靶细胞在同一微流体中 液滴，可以根据目标单元格构建测定法，因为它将沿AB分泌细胞携带 因此，可以在随后的测序步骤中识别AB的RNA。 MOD利用流动 细胞仪式检测和排序，因此很容易扩展HT。这个项目的方法已经 以前的工作在MOD平台上开发测定法。在第一个目标中，将开发两个测定 检测与膜蛋白靶标的AB结合。第一个将采用现有的基于珠子的无洗 与高拷贝数目标一起使用的测定方案，第二个将开发更敏感的测定法 通过清洗步骤低拷贝数目标，并将探索创建耐用物理的适当方法 将通过FACS分类或重新封装的单元格之间的联系。第二个目标将测试和 用来自免疫小鼠的B细胞量化系统，以进行AB发现的现实证明。 B细胞会 使用SARS-COV-2作为抗原，从标准的4周免疫方案中来源 并将用于探索液滴中原代B细胞培养的参数以及其他因素 将B细胞生物学移植到MOD平台上。将测试一个小批次（50-100k）B细胞/靶细胞测定法， 并假设平台的HT将与大量命中率（约1000个阳性测定）相关，则 少数AB候选人的少数（〜10）将在生物信息中选择以后重新连接并击中 验证。成功的MOD启用抗体筛选将引入一个新的范式 研究人员确定一个更大，更多样化的AB候选人领域，绕过当前的成本限制， 可伸缩性，商业可用性或技术复杂性，并最终导致更好的疗法。