Renewable antibodies to secreted proteins and single and multi-pass cell surface

针对分泌蛋白以及单次和多次细胞表面的可再生抗体

• 批准号: 8702409
• 负责人: James D. Marks
• 金额: $ 40.94万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-24 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8702409
• 关键词: Animals; Antibodies; Antibody Therapy; Antigen Targeting; Antigens; Automation; Bacteriophages; Biological Assay; Bypass; Cell Line; Cell Surface Proteins; Cell membrane; Cell surface; Cells; Eukaryotic Cell; Evaluation; Extracellular Protein; Generations; Genes; Goals; Human; Hybridomas; Immune; Immunization; Immunohistochemistry; Immunoprecipitation; In Vitro; Libraries; Mammalian Cell; Membrane Proteins; Methods; Monoclonal Antibodies; N-terminal; Output; Pattern; Phage Display; Proteins; Proteome; Reagent; Recombinants; Research; Research Personnel; Serum; Services; Solutions; Specificity; Surface; Technology; Tertiary Protein Structure; Therapeutic Studies; Time; Tissues; Validation; Western Blotting; Work; Yeasts; base; design; drug discovery; editorial; extracellular; gene cloning; in vivo; in vivo imaging; interest; next generation; polyclonal antibody; protein function; therapeutic target

Antibodies for research and therapy Antibodies (Abs) are essential reagents for determining how proteins function under normal or pathophysiological conditions. Uses include quantifying proteins, identifying the temporal and spatial pattern of expression in cells and tissue, and identifying interacting partners. Such studies require Abs of high specificity that function in assays including Western blotting, immunoprecipitation, immunohistochemistry (IHC) and in vivo imaging. Over half the human proteome is not annotated, and functional Abs are not reliably available for these proteins. Where monoclonal or polyclonal Abs are commercially available, a high proportion show either poor specificity or fail to recognize their targets (1-5). For example, a recent editorial by Michel et al. highlighted the lack of target specificity for 49 Abs against 19 subtypes of GPCRs (6). An additional problem is lot-to-lot variability in Ab specificity, including monoclonal Abs (mAbs) made via hybridoma technology, resulting in inconsistent assay results (4). The purpose of TR&D Project One is to develop high throughput scalable technologies to generate widely available, renewable, validated and standardized sets of Ab reagents (rAbs) to a portion of the secretome consisting of plasma membrane and extracellular proteins. One key aspect of the technology that will be developed is that where possible, the expensive, time consuming and tedious task of antigen generation and purification will be bypassed by displaying the antigen at high levels on the surface of eukaryotic cells, including yeast, and mammalian cell lines. Antigens expressed on mammalian cells or yeast will be used for selection of phage Abs, as well as for validation and characterization. The use of phage display bypasses the low throughput, time consuming, and expensive immunization of animals to generate polyclonal Abs or the use of hybridoma technology to generate mAbs. Moreover, the Ab genes are cloned, the rAbs are forever renewable and can easily be formatted for expression as Ab fragments or traditional mAbs with any Fc. While we will primarily generate rAbs to a subset of the secretome in this Project (the extracellular portions of plasma membrane and extracellular proteins), this approach should be applicable to many or all of the secreted proteins, 20-40% of the proteome.

用于研究和治疗的抗体抗体（Abs）是确定蛋白质在正常或病理生理条件下如何发挥作用的基本试剂。用途包括定量蛋白质，识别细胞和组织中表达的时间和空间模式，以及识别相互作用的伙伴。此类研究需要具有高特异性的抗体，能够在Western blotting、免疫沉淀、免疫组织化学（IHC）和体内成像等检测中发挥作用。超过一半的人类蛋白质组没有被注释，而且这些蛋白质的功能性抗体也不可靠。在市面上可以买到的单克隆或多克隆抗体中，有很大一部分要么特异性差，要么不能识别它们的靶标（1-5）。例如，Michel等人最近的一篇社论强调了49种抗体对19种gpcr亚型缺乏靶向特异性(6)。另一个问题是，包括通过杂交瘤技术制备的单克隆抗体（mab）在内，抗体特异性的批次间可变性导致检测结果不一致(4)。