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(Abbs)是确定蛋白质在正常或病理生理条件下如何发挥作用的重要试剂。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，以及识别相互作用的伙伴。这类研究需要具有高度特异性的抗体，这些抗体可用于免疫印迹、免疫沉淀、免疫组织化学(IHC)和体内成像等检测。超过一半的人类蛋白质组没有注释，这些蛋白质不能可靠地获得功能性抗体。在市场上有单抗或多克隆抗体的地方，高比例的抗体要么显示出较差的特异性，要么无法识别它们的靶标(1-5)。例如，米歇尔等人最近的一篇社论。强调49株抗体缺乏针对19个GPCRs亚型的靶标特异性(6)。另一个问题是抗体特异性的批次到批次的可变性，包括通过杂交瘤技术制造的单抗，导致检测结果不一致(4)。 研发项目一的目的是开发高通量、可扩展的技术，以产生广泛可用的、可再生的、有效的和标准化的抗体试剂(RAB)集，用于由质膜和细胞外蛋白组成的分泌组的一部分。将开发的这项技术的一个关键方面是，在可能的情况下，通过在包括酵母和哺乳动物细胞系在内的真核细胞表面展示高水平的抗原，将绕过昂贵、耗时和乏味的抗原生成和纯化任务。在哺乳动物细胞或酵母上表达的抗原将用于噬菌体抗体的选择以及验证和鉴定。噬菌体展示的使用绕过了低通量、耗时和昂贵的动物免疫来产生多克隆抗体，或者使用杂交瘤技术来产生mAb。此外，抗体基因是克隆的，RAB是永远可再生的，可以很容易地形成抗体片段或具有任何Fc的传统单抗。虽然在这个项目中，我们将主要生成分泌组的一个子集(质膜的胞外部分和胞外蛋白)的RABS，但这种方法应该适用于许多或所有分泌的蛋白质，占蛋白质组的20%-40%。