Droplet Compartmentalized Selection for Deep-Mining of Antibody Diversity

抗体多样性深度挖掘的液滴区室化选择

• 批准号: 7672772
• 负责人: JOHN A HEYMAN
• 金额: $ 9.97万
• 依托单位: HABSEL, INC.
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-01 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7672772
• 关键词: Affinity; Antibodies; Antibody Diversity; Antibody Formation; Antibody-Producing Cells; Antigens; Apoptosis; Binding; Biological Assay; Blood; Bypass; Calibration; Cell Line; Cell Separation; Cell Survival; Cells; Characteristics; Color; Computer Systems Development; Diagnostic tests; Diagnostics Research; Drops; Encapsulated; Equilibrium; Evaluation; Floor; Flow Cytometry; Fluorescein-5-isothiocyanate; Generations; Genes; Hour; Human; Hybridomas; Immune; Immunoglobulin-Secreting Cells; Individual; Label; Lasers; Marketing; Measurement; Measures; Methods; Microfluidics; Mining; Molecular Target; Monoclonal Antibodies; Operative Surgical Procedures; Optics; Oryctolagus cuniculus; Output; Patients; Pharmacologic Substance; Phase; Phase I Clinical Trials; Phase II Clinical Trials; Plasma Cells; Population; Process; Production; Reading; Recovery; Relative (related person); Reverse Transcriptase Polymerase Chain Reaction; Screening procedure; Sorting - Cell Movement; Source; Specificity; Speed; Stream; System; Techniques; Technology; Testing; Therapeutic Monoclonal Antibodies; Therapeutic antibodies; Time; Variant; Work; antigen binding; base; cancer therapy; carcinogenesis; cell immortalization; cell type; cyanine dye 5; expression vector; high throughput screening; immortalized cell; interest; novel diagnostics; prevent; processing speed; public health relevance; research study; therapy design; tool; tumor growth

DESCRIPTION (provided by applicant): We seek to develop a completely new microfluidics-based method (Droplet Compartmentalized Selection - DCS) to isolate cells that produce antibodies with desired binding characteristics. In DCS, individual cells are encapsulated (together with components to assay binding activity) in pico-liter scale droplets, which can then be screened for binding activity at rates of about 1,000 droplets per second. Individual cells will fill the droplets with antibody to (M concentrations within a few hours. A fluorescent read-out can then be used to identify and collect droplets that contain the desired antibody-producing cells. Crucially, there is no need to immortalize the antibody-producing cells. Instead, the antibody-encoding genes can be isolated from individual selected cells by reverse-transcriptase PCR and then cloned into appropriate expression vectors. DCS has three major advantages over typical methods to isolate monoclonal antibodies. 1) Because the screening can be performed on individual, non-immortalized cells, a great deal of time is saved. There is no need to create hybridomas to immortalize the antibody-producing cells, and the time required to grow and assay individual "clones" is several hours rather than several weeks. 2) DCS allows access to much greater antibody diversity. By eliminating the highly inefficient process of hybridoma generation (typically only 1/100,000 antibody-producing cells are immortalized), and by greatly speeding the screening process, it is possible to screen tens- or hundreds- of thousands of individual cells for the ability to produce desired antibodies. In contrast, a typical monoclonal antibody screen can analyze at most several hundred unique antibody-producing hybridomas. And 3), DCS will allow screening of cell types which are not amenable to typical hybridoma-based methods. For example, cells that produce antibodies against particular targets could be isolated from blood from human patients. This could pave the way for novel diagnostic tests and would facilitate identification of endogenous human antibodies that might serve as building blocks for therapeutic antibodies. PUBLIC HEALTH RELEVANCE: Therapeutic monoclonal antibodies make up the fastest-growing segment of the prescription pharmaceutical market and are the cornerstone of an emerging class of 'targeted' cancer therapies that are designed to prevent tumor growth by more specific actions than older treatments, via the targeting of molecular drivers of carcinogenesis. Our new microfluidics-based method (Droplet Compartmentalized Selection - DCS) to isolate cells that produce antibodies with desired binding characteristics will revolutionize the production of mAbs. It will enable much higher throughput screening, giving far greater access to immune repertoires, thus allowing the isolation of more and better antibody therapeutics. Moreover, because the antibodies from a single cell can be detected, it becomes possible to directly screen non-immortalized cells instead of hybridomas, further increasing the antibody diversity that can be accessed.

描述（由申请人提供）：我们寻求开发一种全新的基于微流体的方法（液滴区室化选择- DCS），以分离产生具有所需结合特征的抗体的细胞。在DCS中，单个细胞被封装（与测定结合活性的组分一起）在皮升规模的液滴中，然后可以以每秒约1，000个液滴的速率筛选结合活性。单个细胞将在几小时内用抗体填充液滴以达到0 M浓度。然后可以使用荧光读出来识别和收集含有所需抗体产生细胞的液滴。最重要的是，不需要使抗体产生细胞永生化。相反，可以通过逆转录酶PCR从各个选择的细胞中分离抗体编码基因，然后克隆到适当的表达载体中。DCS与分离单克隆抗体的典型方法相比有三个主要优点。1)因为筛选可以在单个的非永生化细胞上进行，所以节省了大量的时间。不需要创建杂交瘤来使产生抗体的细胞永生化，并且生长和测定单个“克隆”所需的时间是几个小时而不是几周。2)DCS允许获得更大的抗体多样性。通过消除杂交瘤产生的非常低效的过程（通常只有1/100，000的抗体产生细胞是永生化的），并且通过大大加速筛选过程，可以筛选数万或数十万个具有产生所需抗体的能力的单个细胞。相比之下，典型的单克隆抗体筛选最多可以分析几百个独特的产生抗体的杂交瘤。和3），DCS将允许筛选不适合典型的基于杂交瘤的方法的细胞类型。例如，可以从人类患者的血液中分离出产生针对特定靶标的抗体的细胞。这可能为新的诊断测试铺平道路，并将有助于鉴定可能作为治疗性抗体构建模块的内源性人类抗体。 公共卫生关系：治疗性单克隆抗体构成了处方药市场增长最快的部分，并且是新兴的一类“靶向”癌症治疗的基石，这些治疗旨在通过比旧治疗更具体的行动来预防肿瘤生长，通过靶向致癌的分子驱动因素。我们新的基于微流体的方法（液滴区室化选择- DCS）分离产生具有所需结合特性的抗体的细胞，将彻底改变mAb的生产。它将实现更高的通量筛选，使更多的免疫库，从而允许更多和更好的抗体治疗剂的分离。此外，由于可以检测来自单个细胞的抗体，因此可以直接筛选非永生化细胞而不是杂交瘤，进一步增加了可以获得的抗体多样性。