A high throughput pipeline to select renewable recombinant polyclonal antibodies

这项应用的目标是建立一条试点的、高通量的三级抗体选择流水线。一级抗体将是可再生的重组多克隆抗体，可从1毫升扩增至100,000升，而不会损失多样性。最终，它们将根据所有人类蛋白质进行筛选。在结构上，提供的抗体将与传统抗体相似，允许科学家在不修改方案的情况下使用它们。然而，与传统的多克隆不同，它们将类似于亲和纯化的多克隆，大多数抗体都指向感兴趣的目标。这些一级多克隆细胞应该能满足大多数研究需要。如有需要，将从第一级多克隆抗体库中分离出第二级单抗。第三级单抗将是定制项目，需要从第二级抗体引线创建衍生库，并选择具有更高亲和力或特异性的单克隆。针对所有人类蛋白的抗体的广泛应用将促进对人类疾病的理解，并为治疗干预提供可能的靶点，抗体本身有可能成为治疗线索。前两个具体目标涉及为目标抗原和抗体库生产建立质量控制管道；第三个目标将这些结合在抗体选择管道中，而在第四个目标中，选定的抗体被表征。该项目将结合噬菌体和酵母抗体展示，从噬菌体抗体库中进行初始选择，并从通过将噬菌体输出克隆到酵母展示载体中创建的酵母库中进行后续选择。多克隆池将被表达为单基因ScFv-Fc融合，使用进化为高表达水平的小鼠或兔Fc结构域。在内部抗体验证之后，将在人类蛋白质图谱上进行鉴定，该图谱有一个广泛的管道，可以表征抗体的特异性、组织和细胞分布。与针对每个目标选择和鉴定单克隆相比，这种三层方法将使吞吐量增加约100倍，从而显著降低成本。可再生的多克隆细胞将具有很高的功能，并在需要时为单克隆细胞提供来源。

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.