Isolation of functional IgGs in the cytoplasm of a novel E. coli expression host

描述（由申请人提供）：单克隆抗体（mab）在人类健康方面具有很大的前景，其应用范围从靶向癌细胞的治疗剂到可以检测给定抗原痕量水平的诊断性生物标志物。抗体的全球销售额在2007年达到近310亿美元，预计到2012年未来销售额将达到560亿美元，复合年增长率为13%。推动这种快速增长的是重组DNA技术，它直接导致了一些强大技术的发展，这些技术被广泛用于设计人类单克隆抗体和抗体衍生片段，这些片段对几乎任何目标抗原都具有高亲和力和特异性。从治疗的角度来看，全长单克隆抗体或igg往往优于较小的抗体片段，因为它们在哺乳动物体内的循环半衰期较长，这是由于它们的大分子大小阻止了肾脏的清除，并且它们能够通过使用挽救途径避免内皮细胞中的蛋白质水解。然而，由于这些多亚基蛋白的复杂性，它们的生产在很大程度上局限于真核表达系统，如CHO或杂交瘤细胞，因此繁琐、昂贵、耗时，并且不适合并行化。由于这些缺点，现有的发现和生产igg的技术一直在努力跟上对这些重要生物分子快速增长的需求。为了帮助弥补与IgG生产相关的技术差距，大肠杆菌细胞因其简单、生长速度快、易于使用和成本低而成为一种有吸引力的选择。然而，虽然大肠杆菌已被证明是表达较小抗体片段（如Fvs、scFvs、fab或F（ab’）2s）的良好宿主，但其表达IgG和工程的潜力尚未得到充分研究。因此，本提案的目标是开发大肠杆菌作为发现、工程和制造全长人igg的强大载体。在Specific Aim 1下，一种新的大肠杆菌菌株将被设计成专门针对重组igg在细胞质室中的高水平表达。本提案的特定目标2旨在开发一种独特的筛选方法，用于直接选择“细胞克隆物”-从活的大肠杆菌细胞的细胞质中分离出的给定蛋白质抗原的功能性igg。该筛选将基于广泛用于检测蛋白质-蛋白质相互作用的流行的分裂-蛋白质系统。该筛选的实用性将通过筛选针对目标蛋白抗原的细胞克隆IgG序列的大型合成文库来证明。与大多数其他抗体选择-表达系统不同，所提出的策略是一种独特的分析开发、文库设计和宿主细胞工程的整合。这些研究的成功完成将极大地扩展可用于生产和设计不同抗原特异性的全长igg的工具箱，这些igg可用于基础研究、诊断和治疗。

DESCRIPTION (provided by applicant): Monoclonal antibodies (mAbs) hold great promise in human health with applications ranging from therapeutic agents that target cancer cells, to diagnostic biomarkers that can detect trace levels of a given antigen. This promise is best reflected in global sales of antibodies which reached nearly $31 billion in 2007 and future sales predicted to reach $56 billion by 2012, a compound annual growth rate of 13%. Stoking this rapid growth is recombinant DNA technology, which has led directly to the development of a handful of powerful technologies that are widely exploited to engineer human mAbs and antibody-derived fragments with high affinity and specificity for virtually any target antigen. From a therapeutic standpoint, full-length mAbs or IgGs are often advantageous over smaller antibody fragments due to their long circulating half-life in mammals, which results from a combination of their large molecular size that prevents clearance in the kidneys and their ability to avoid proteolysis in the endothelium by using a salvage pathway. However, due to the complexity of these multi- subunit proteins, their production has largely been restricted to eukaryotic expression systems such as CHO or hybridoma cells and is therefore cumbersome, expensive, time consuming, and not amenable to parallelization. As a result of these shortcomings, the existing technologies for discovery and production of IgGs have struggled to keep pace with the rapidly growing demand for these important biomolecules. To help bridge the technological gap associated with IgG production, Escherichia coli cells represent an attractive option due to their simplicity, rapid growth rate, ease of use and low cost of goods. However, while E. coli has proven to be an excellent host for the expression of smaller antibody fragments such as Fvs, scFvs, Fabs or F(ab')2s, its potential for IgG expression and engineering has not been thoroughly investigated. Therefore, the goal of this proposal is to develop E. coli as a robust vehicle for the discovery, engineering and manufacturing of full-length human IgGs. Under Specific Aim 1, a novel E. coli strain will be engineered that is specifically geared towards high-level expression of recombinant IgGs in the cytoplasmic compartment. Specific Aim 2 of this proposal seeks to develop a unique screening method for direct selection of "cytoclonals" - functional IgGs to a given protein antigen isolated from the cytoplasm of living E. coli cells. This screen will be based on the popular split-protein system widely used for detecting protein-protein interactions. The utility of this screen will be demonstrated by screening a large synthetic library of IgG sequences for cytoclonals against target protein antigens. Unlike most other antibody selection-expression systems, the proposed strategy is a unique integration of assay development, library design, and host cell engineering. Successful completion of these studies will greatly expand the toolkit available for producing and engineering full-length IgGs of different antigen specificities that can be used in basic research, diagnosis and therapy. PUBLIC HEALTH RELEVANCE: Monoclonal antibodies and antibody-based fragments account for >30% of all revenues in the biotechnology market and are used to treat a wide array of human diseases including asthma, autoimmune diseases, bacterial and viral infections, cancer and other diseases. Since antibody therapies are an increasingly large fraction of the drugs in development, with ever escalating increases in the cost of drug development, any improvements to the production or discovery of efficacious antibodies will have a significant impact on human health. Accordingly, this proposal seeks to develop Escherichia coli cells as a technology platform for rapid, low-cost expression and isolation of full-length human monoclonal antibodies against virtually any target protein antigen of interest.