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.

研究和治疗抗体（ABS）的抗体是确定蛋白质在正常或病理生理状况下的作用的重要试剂。用途包括量化蛋白质，识别细胞和组织中表达的时间和空间模式，并识别相互作用的伴侣。这样的研究需要具有高特异性的ABS，即在包括蛋白质印迹，免疫沉淀，免疫组织化学（IHC）和体内成像在内的测定中起作用。超过一半的人蛋白质组没有注释，并且这些蛋白质无法可靠地使用功能性ABS。如果单克隆或多克隆ABS可商购，则高比例表明特异性较差或无法识别其靶标（1-5）。例如，Michel等人最近的社论。强调了49次ABS的目标特异性与19个亚型的GPCR（6）。另一个问题是AB特异性的大量变化，包括通过杂交瘤技术制造的单克隆ABS（mAb），从而导致不一致的测定结果（4）。 TR＆D项目ONE的目的是开发高吞吐量可扩展技术，以生成广泛可用的，可再生的，经过验证和标准化的AB试剂集（RAB），以将质膜和细胞外蛋白质组成的一部分。将要开发的技术的一个关键方面是，抗原产生和纯化的昂贵，耗时和乏味的任务将通过在真核细胞表面高水平（包括酵母和哺乳动物细胞系）表面上显示抗原来绕开。在哺乳动物细胞或酵母上表达的抗原将用于选择噬菌体ABS，以及验证和表征。噬菌体展示的使用绕过了动物的低吞吐量，耗时和昂贵的免疫，以产生多克隆ABS或使用杂交瘤技术产生mAb。此外，AB基因是克隆的，Rabs永远可再生，并且可以轻松地格式化，以表达AB片段或带有任何FC的传统mAb。尽管我们将主要在该项目（质膜和细胞外蛋白的细胞外部分）中生成RABS，但该方法应适用于许多或所有分泌的蛋白质，占蛋白质组的20-40％。