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

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功能的必要试剂。用途包括定量蛋白质，鉴定细胞和组织中表达的时间和空间模式，以及鉴定相互作用的伴侣。这些研究需要在包括蛋白质印迹、免疫沉淀、免疫组织化学（IHC）和体内成像的测定中起作用的高特异性Ab。超过一半的人类蛋白质组没有注释，并且这些蛋白质的功能性抗体不可靠。当单克隆或多克隆抗体可商购获得时，高比例的抗体显示出较差的特异性或不能识别其靶标（1-5）。例如，Michel等人最近的一篇社论强调了49种Ab对19种GPCR亚型缺乏靶特异性（6）。另一个问题是Ab特异性的批间变异性，包括通过杂交瘤技术制备的单克隆抗体（mAb），导致测定结果不一致（4）。 TR&D项目一的目的是开发高通量可扩展技术，以产生广泛可用的，可再生的，经验证的和标准化的抗体试剂（rAb），用于由质膜和细胞外蛋白组成的分泌组的一部分。将要开发的技术的一个关键方面是，在可能的情况下，通过在真核细胞（包括酵母和哺乳动物细胞系）的表面上以高水平展示抗原，将绕过抗原产生和纯化的昂贵、耗时和繁琐的任务。在哺乳动物细胞或酵母上表达的抗原将用于噬菌体Ab的选择，以及用于验证和表征。噬菌体展示的使用绕过了低通量、耗时和昂贵的动物免疫以产生多克隆Ab或使用杂交瘤技术来产生mAb。此外，克隆Ab基因，rAb是永远可再生的，并且可以容易地格式化为Ab片段或具有任何Fc的传统mAb用于表达。虽然我们将主要在本项目中产生针对分泌组亚组的rAb（质膜的细胞外部分和细胞外蛋白），但这种方法应适用于许多或所有分泌蛋白，占蛋白质组的20-40%。

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.