High-throughput antibody discovery directly from B cells using nanovial technology

使用纳米瓶技术直接从 B 细胞发现高通量抗体

• 批准号: 10324363
• 负责人: Joseph de Rutte
• 金额: $ 35.51万
• 依托单位: PARTILLION BIOSCIENCE CORPORATION
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-16 至 2023-01-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10324363
• 关键词: Address; Adopted; Adoption; Affinity; Alpha Particles; Antibodies; Antibody Affinity; Antibody Diversity; Antibody Repertoire; Antigen Targeting; Antigens; Award; B-Lymphocytes; Back; Binding; Biological Assay; Capital; Cell Separation; Cell surface; Cells; Centrifugation; Clonal Expansion; Data; Development; Diagnostic tests; Dose; Drops; Enzyme-Linked Immunosorbent Assay; Equipment; Event; Fluorescence-Activated Cell Sorting; Foundations; Hybridomas; Hydrogels; Immunize; Individual; Lead; Light; Link; Measures; Methods; Microfluidics; Monoclonal Antibodies; Monoclonal Antibody Therapy; Mus; Patients; Pharmaceutical Preparations; Phase; Population; Process; Property; Reagent; Recovery; Running; Site; Small Business Innovation Research Grant; Sorting - Cell Movement; Surface; Technology; Tetanus Toxoid; Time; Work; anti-IgG; antibody diagnostic; base; bioinformatics pipeline; cost; fluorescence activated cell sorter device; improved; indexing; innovation; instrument; mRNA sequencing; nanolitre; prevent; response; screening; side effect; single cell mRNA sequencing; single cell sequencing; targeted treatment

ABSTRACT Six out of ten top-grossing drugs in 2019 were monoclonal antibodies (mAbs), up from zero in 2010. By enabling highly-targeted therapies with enhanced efficacy and reduced side effects, mAbs have provided extraordinary benefits to patients. However, the discovery of mAb therapies remain slow and laborious owing to limited ability to quickly find antibodies with optimal functional properties (e.g., affinity). Most widely used methods still rely on immortalization of fragile B cells into hybridomas, followed by clonal expansion, ELISA screening, sequencing, and transient expression for downstream analysis of function. This entire process can take months and suffers from significant loss in the diversity of antibody sequences due to the immortalization process and the limited throughput of the standard well-plate formats. Emerging automated microfluidic workflows enable direct screening of B cells, improving diversity, but are still limited in the number of B cells screened. These specialized instruments are also not widely available, partly due to high capital equipment and consumable costs. Thus there is a critical need for a high-throughput single-cell screening workflow that does not rely on new specialized capital equipment and still maintains a linkage between antibody function and antibody sequence. To address these limitations, we will develop a lab on a particle-enabled workflow that enables customers to (i) select B cells secreting high affinity antibodies using standard fluorescence activated cell sorters (FACS) and (ii) link antibody function directly with paired heavy and light chain sequences in individual B cells. Our technology is based on microscale crescent-shaped hydrogel nanovials which capture cells, are functionalized to capture secretions, and enable formation of millions of uniform nanoliter droplets to prevent the loss and cross-talk of secretions. Like for standard ELISAs in microwells, nanovials can be exchanged between multiple solutions enabling functional assays of captured secreted antibodies, while maintaining the linkage of this information with the attached cells. In this Phase I SBIR, we will develop workflows to measure affinity of antibodies secreted by individual cells to a target antigen using an on-nanovial sandwich assay. We will use this assay to screen individual B cells from mice immunized against tetanus toxoid based on affinity using FACS and perform downstream single cell sequencing to identify matched heavy and light chain sequences. We will validate this process by transiently expressing identified VH and VL pairs with different measured affinities to compare with bulk ELISA. At the completion of the proposed work we will have demonstrated the ability to screen over 2 million B cells based on secreted antibody affinity in a single run, and identify potential antibody leads in <1 week. Following the successful completion of our aims we will have laid a strong foundation for an easily adopted reagent product that would provide significantly more antibody repertoire depth and the ability to select based on functional properties at early stages of discovery, ultimately reducing time and costs for antibody developers.

抽象的 2019年，十种最高的药物中有六种是单克隆抗体（mAb），从2010年的零上升。 MAB提供了高度靶向的疗法，具有增强的疗效和副作用，提供了非凡的疗法 对患者的好处。但是，由于能力有限，MAB疗法的发现仍然缓慢而费力 快速找到具有最佳功能特性（例如亲和力）的抗体。最广泛使用的方法仍然依赖 将脆弱的B细胞永生化成杂交瘤，然后再膨胀，ELISA筛查，测序， 和瞬态表达以进行功能下游分析。整个过程可能需要几个月的时间，并且会遭受痛苦 由于永生过程而导致的抗体序列多样性的显着丧失和有限 标准设备格式的吞吐量。新兴的自动微流体工作流程启用直接 筛选B细胞，改善多样性，但在筛选的B细胞数量中仍然受到限制。这些专业 仪器也不广泛可用，部分是由于高资本设备和可消耗成本所致。因此 对于不依赖新的专业资本的高通量单细胞筛选工作流程是至关重要的 设备并仍然保持抗体功能和抗体序列之间的联系。解决这些 局限性，我们将在支持粒子的工作流程上开发一个实验室，使客户能够（i）选择B单元格 使用标准荧光激活的细胞分角（FACS）和（ii）链接抗体分泌高亲和力 直接在单个B细胞中成对的重链和轻链序列发挥作用。我们的技术基于 捕获细胞的显微镜新月形水凝胶纳米凝胶纳米腔，可捕获分泌物，以捕获分泌物， 并能够形成数百万个均匀的纳米液滴，以防止分泌物的损失和串扰。 就像微孔中的标准ELISA一样，可以在多种解决方案之间交换纳米维体 捕获的分泌抗体的功能测定，同时保持此信息与 附着的细胞。在此阶段I SBIR中，我们将开发工作流程以测量由分泌的抗体的亲和力 单个细胞使用纳维三明治测定法到靶抗原。我们将使用此测定法进行筛选 基于FACS的亲和力，来自针对破伤风毒素的小鼠的单个B细胞，并执行 下游单细胞测序以识别匹配的重链序列。我们将验证这个 通过瞬时表达具有不同测量亲和力的VH和VL对的过程 散装Elisa。拟议的工作完成时，我们将证明能够筛选超过200万 B细胞基于单个运行中的分泌抗体亲和力，并鉴定潜在的抗体在<1周内导致。 成功完成目标之后，我们将为轻松采用的坚实基础 试剂产品将提供更多的抗体曲目深度和选择基于的抗体曲目。 关于发现早期阶段的功能性能，最终减少了抗体开发人员的时间和成本。