Droplet Compartmentalized Selection for Deep-Mining of Antibody Diversity

描述(申请人提供)：我们寻求开发一种全新的基于微流控技术的方法(液滴分隔选择)来分离产生具有所需结合特性的抗体的细胞。在分布式控制系统中，单个细胞被包裹在皮升尺度的液滴中(连同成分以测定结合活性)，然后可以大约每秒1000个液滴的速度筛选结合活性。个别细胞将在几个小时内用M浓度的抗体填充液滴。然后，可以使用荧光读数来识别和收集包含所需抗体产生细胞的液滴。最重要的是，没有必要使产生抗体的细胞永生。相反，可以通过逆转录-聚合酶链式反应从单个选定的细胞中分离抗体编码基因，然后将其克隆到适当的表达载体中。与传统的单抗分离方法相比，DSC有三个主要优点。1)由于可以对单个的非永生化细胞进行筛选，因此节省了大量的时间。没有必要创造杂交瘤来使产生抗体的细胞永生化，培养和检测单个“克隆”所需的时间是几个小时，而不是几个星期。2)DCs允许获得更大的抗体多样性。通过消除杂交瘤生成效率极低的过程(通常只有1/10万产生抗体的细胞永生化)，并通过极大地加快筛选过程，有可能对数万个--甚至数十万--单个细胞进行筛选，以获得产生所需抗体的能力。相比之下，一个典型的单抗筛查最多可以分析数百个产生抗体的独特杂交瘤。和3)，分散控制系统将允许筛选不适用于典型杂交瘤方法的细胞类型。例如，可以从人类患者的血液中分离出产生针对特定目标的抗体的细胞。这可能为新的诊断测试铺平道路，并将有助于识别内源性人类抗体，这些抗体可能成为治疗性抗体的基石。 与公共健康相关：治疗性单抗是处方药市场增长最快的部分，也是一种新兴的“靶向”癌症疗法的基石，这种疗法旨在通过靶向致癌的分子驱动因素，通过比旧疗法更具体的行动来防止肿瘤生长。我们以微流控为基础的新方法(液滴隔室选择--DC)分离产生具有所需结合特性的抗体的细胞，将彻底改变单抗的生产。它将实现更高的吞吐量筛查，使更多的人能够获得免疫谱系，从而能够分离出更多和更好的抗体疗法。此外，由于可以检测到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，从而进一步增加了可获得的抗体多样性。

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.