A high throughput pipeline to select renewable recombinant polyclonal antibodies

本申请的目的是建立一个试点，高通量，三层抗体选择管道。一级抗体将是可再生的重组多克隆抗体，可以从1ml扩增到>/=100,000升而不损失多样性。最终，它们将被选中对抗所有人类蛋白质。在结构上，提供的抗体与传统抗体相似，允许科学家在不修改方案的情况下使用它们。然而，与传统的多克隆物不同，它们类似于亲和纯化的多克隆物，大多数抗体指向感兴趣的目标。这些一级多克隆应该满足大多数研究需求。如有需要，将从第一级多克隆抗体池中分离第二级单克隆抗体。第三层单克隆抗体将是定制项目，需要从第二层抗体先导物中创建衍生文库，并选择具有改进亲和力或特异性的单克隆抗体。针对所有人类蛋白质的抗体的广泛可用性将促进对人类疾病的了解，并为治疗干预提供可能的目标，抗体本身具有成为治疗线索的潜力。前两个具体目标涉及建立目标抗原和抗体库生产的质量控制管道；第三种方法将这些结合在抗体选择管道中，而在第四种方法中，选定的抗体被表征。该项目将结合噬菌体和酵母抗体展示，最初从噬菌体抗体文库中进行选择，随后从通过将噬菌体输出克隆到酵母展示载体中创建的酵母文库中进行选择。多克隆池将表达为单基因scFv-Fc融合物，使用进化为高表达水平的小鼠或兔Fc结构域。在内部抗体验证后，将在人类蛋白质图谱上进行表征，该图谱具有广泛的管道来表征抗体的特异性、组织和细胞分布。与针对每个靶点的单克隆选择和表征相比，这种三层方法将使通量提高约100倍，从而显著降低成本。可再生的多克隆克隆具有很高的功能，并在需要时为单克隆克隆提供了来源。

The objective of this application is to establish a pilot, high-throughput, three-tiered antibody selection pipeline. Tier one antibodies will be renewable recombinant polyclonal antibodies that can be amplified from 1 ml to >/=100,000 liters with no loss of diversity. Eventually they will be selected against all human proteins. Structurally, the provided antibodies will be similar to traditional antibodies, allowing scientists to use them without modifying protocols. However, unlike traditional polyclonals, they will resemble affinity-purified polyclonals, with most antibodies directed towards the target of interest. These tier one polyclonals should satisfy most research needs. Where required, second tier monoclonal antibodies will be isolated from first tier polyclonal antibody pools. Third tier monoclonal antibodies will be custom projects requiring the creation of derivative libraries from second tier antibody leads, and the selection of monoclonals with improved affinities or specificities. The broad availability of antibodies against all human proteins will facilitate the understanding of human disease, and provide likely targets for therapeutic intervention, with the antibodies themselves having the potential to become therapeutic leads. The first two specific aims involve the creation of quality control pipelines for target antigen and antibody library production; the third combines these in the antibody selection pipeline, while in the fourth, selected antibodies are characterized. This project will combine phage and yeast antibody display, with initial selections carried out from phage antibody libraries, and subsequent selections from yeast libraries created by cloning phage outputs into yeast display vectors. Polyclonal pools will be expressed as single gene scFv-Fc fusions, using murine or rabbit Fc domains evolved for high expression levels. After internal antibody validation, characterization will be carried out at the Human Protein Atlas, which has an extensive pipeline characterizing antibodies for specificity, tissue and cellular distribution. This three-tiered approach will increase throughput by ~100 fold compared to the selection and characterization of monoclonals against each target, resulting in a dramatic reduction in costs. The renewable polyclonals will be highly functional, and provide a source for monoclonals when needed.