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A high throughput pipeline to select renewable recombinant polyclonal antibodies

选择可再生重组多克隆抗体的高通量管道

基本信息

批准号：
8335448
负责人：
ANDREW BRADBURY
金额：
$ 141.1万
依托单位：
LOS ALAMOS NAT SECTY-LOS ALAMOS NAT LAB
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-09-25 至 2014-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8335448
关键词：
Affinity Antibodies Antibody Diversity Antibody Specificity Antigen Targeting Atlases Bacteriophages Binding Cloning Coupled Custom Dementia Diabetes Mellitus Ensure Fc domain Flow Cytometry Gene Targeting Generations Generic Drugs Genes Heart Diseases Human Immunofluorescence Immunologic Immunohistochemistry In Situ Laboratories Letters Libraries Malignant Neoplasms Management Information Systems Methods Molecular Weight Monoclonal Antibodies Mus Oryctolagus cuniculus Output Process Production Protein Array Protein Fragment Proteins Protocols documentation Quality Control Recombinants Reporter Research Role Scientist Sequence Analysis Signal Transduction Solubility Sorting - Cell Movement Source Specificity Therapeutic Therapeutic Intervention Tissues Validation Western Blotting Yeasts base cost human disease improved interest novel polyclonal antibody polypeptide protein complex scaffold structural genomics tool vector

项目摘要

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.

该应用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 1 ml 扩增至 >/= 100,000 升，而不会损失多样性。最终它们将在所有人类蛋白质中被选择。从结构上讲，所提供的抗体将与传统抗体相似，允许科学家在不修改实验方案的情况下使用它们。然而，与传统的多克隆抗体不同，它们类似于亲和纯化的多克隆抗体，大多数抗体针对感兴趣的靶标。这些一级多克隆应该可以满足大多数研究需求。如果需要，将从第一层多克隆抗体池中分离出第二层单克隆抗体。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创建衍生文库，并选择具有改进的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有助于对人类疾病的理解，并为治疗干预提供可能的目标，而抗体本身有可能成为治疗先导药物。前两个具体目标涉及为目标抗原和抗体库生产创建质量控制管道；第三个将这些结合到抗体选择流程中，而第四个则对选定的抗体进行表征。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，以及通过将噬菌体输出克隆到酵母展示载体中而创建的酵母库进行后续选择。多克隆池将表达为单基因 scFv-Fc 融合体，使用为高表达水平而进化的鼠或兔 Fc 结构域。内部抗体验证后，将在人类蛋白质图谱上进行表征，该图谱拥有广泛的管道来表征抗体的特异性、组织和细胞分布。与针对每个靶标的单克隆抗体的选择和表征相比，这种三层方法将通量提高约 100 倍，从而大幅降低成本。可再生的多克隆抗体将具有很高的功能，并在需要时提供单克隆抗体的来源。