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：
8533069
负责人：
ANDREW BRADBURY
金额：
$ 10万
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/8533069
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扩增到>/= 100,000升，而不会损失多样性。最终，它们将被选为所有人类蛋白质。从结构上讲，所提供的抗体将类似于传统抗体，使科学家可以在不修改方案的情况下使用它们。但是，与传统的多克隆不同，它们将类似于亲和纯化的多克隆，大多数抗体针对感兴趣的靶标。这些一级多克隆应满足大多数研究需求。在需要的情况下，将从第一层多克隆抗体库中分离第二层单克隆抗体。第三层单克隆抗体将是自定义项目，需要从第二层抗体铅创建衍生文库，以及选择具有改善亲和力或特异性的单克隆群体。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理解，并为治疗干预提供了可能的靶标，抗体本身具有成为治疗性铅的潜力。前两个具体目的涉及创建用于靶抗原和抗体库生产的质量控制管道。第三个将这些结合在抗体选择管道中，而在第四个中，则表征了选定的抗体。该项目将结合噬菌体和酵母抗体的显示，并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显示载体创建的酵母库中的后续选择。多克隆池将使用以高表达水平演变的鼠或兔FC结构域表示为单基因SCFV-FC融合。在内部抗体验证后，将在人类蛋白质图集进行表征，该图表具有广泛的管道，该管道表征了特异性，组织和细胞分布的抗体。与对每个目标的单克隆的选择和表征相比，这种三层方法将使吞吐量增加约100倍，从而大大降低成本。可再生多克隆将具有高度功能，并在需要时为单克隆人提供源。