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

在新型大肠杆菌表达宿主的细胞质中分离功能性 IgG

• 批准号: 8200628
• 负责人: Mehmet Berkmen
• 金额: $ 21.34万
• 依托单位: NEW ENGLAND BIOLABS, INC.
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-30 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8200628
• 关键词: Accounting; Affinity; Antibodies; Antibody Therapy; Antigen Targeting; Antigens; Asthma; Autoimmune Diseases; Avidity; B-Lymphocytes; Back; Bacterial Infections; Basic Science; Binding; Binding Proteins; Biochemical Process; Biological Assay; Biological Markers; Biotechnology; Bypass; Capital; Cell Surface Proteins; Cell surface; Cells; Chromosomes; Cytoplasm; Cytoplasmic Protein; Cytosine; Development; Diagnosis; Diagnostic; Disease; Endothelium; Engineering; Escherichia coli; Exhibits; Fc domain; Fermentation; Future; Genes; Genetic Engineering; Goals; Growth; Half-Life; Health; Human; Hybridomas; Immunoglobulin Fragments; Immunoglobulin G; Immunoglobulins; Kidney; Length; Libraries; Life; Link; Malignant Neoplasms; Mammalian Cell; Mammals; Marketing; Methods; Monoclonal Antibodies; Noise; Pathway interactions; Phage Display; Pharmaceutical Preparations; Plasmids; Process; Production; Property; Protein Fragment; Protein Subunits; Proteins; Proteolysis; Recombinants; Reporter; Reporting; Ribosomal RNA; Sales; Screening procedure; Serum; Signal Transduction; Specificity; Structure; System; Technology; Tertiary Protein Structure; Testing; Therapeutic; Therapeutic Agents; Time; Transgenic Mice; Treatment Efficacy; Viral; Virus Diseases; antigen binding; assay development; base; cancer cell; cellular engineering; combinatorial; cost; design; disulfide bond; drug development; extracellular; glycosylation; human disease; human monoclonal antibodies; inorganic phosphate; interest; microorganism; molecular size; novel; periplasm; prevent; promoter; protein expression; protein protein interaction; rapid growth; therapeutic development; therapeutic protein; tool; yeast protein

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.

描述（由申请人提供）：单克隆抗体（mAb）在人类健康中具有很大的前景，其应用范围从靶向癌细胞的治疗剂到可以检测给定抗原的痕量水平的诊断生物标志物。这一承诺最好地反映在全球抗体销售额中，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工程化人类mAb和抗体衍生片段，对几乎任何靶抗原都具有高亲和力和特异性。从治疗的角度来看，全长mAb或IgG通常优于较小的抗体片段，这是由于它们在哺乳动物中的长循环半衰期，这是由于它们的大分子尺寸（其阻止肾脏中的清除）和它们通过使用补救途径避免内皮中的蛋白水解的能力的组合。然而，由于这些多亚基蛋白质的复杂性，它们的生产在很大程度上限于真核表达系统，如CHO或杂交瘤细胞，因此是麻烦、昂贵、耗时的，并且不适合并行化。由于这些缺点，用于发现和生产IgG的现有技术一直难以跟上对这些重要生物分子的快速增长的需求。为了帮助弥合与IgG生产相关的技术差距，大肠杆菌细胞由于其简单性、快速生长速度、易用性和低成本而成为一种有吸引力的选择。然而，E.大肠杆菌已被证明是表达较小抗体片段如Fv、scFv、Fab或F（ab '）2s的优良宿主，其用于IgG表达和工程化的潜力尚未被彻底研究。因此，本提案的目标是开发E。大肠杆菌作为一个强大的工具，用于发现，工程和全长人IgG的生产。在Specific Aim 1下，提出了一种新的E.大肠杆菌菌株将被工程化，其特别适合于重组IgG在细胞质区室中的高水平表达。该建议的具体目标2寻求开发一种独特的筛选方法，用于直接选择从活的大肠杆菌细胞质中分离的给定蛋白抗原的“细胞克隆”功能性IgG。coli细胞。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。该筛选的效用将通过筛选针对靶蛋白抗原的细胞克隆的IgG序列的大合成文库来证明。与大多数其他抗体选择表达系统不同，所提出的策略是检测开发、文库设计和宿主细胞工程的独特整合。这些研究的成功完成将大大扩展可用于生产和工程化具有不同抗原特异性的全长IgG的工具包，这些IgG可用于基础研究、诊断和治疗。 公共卫生关系：单克隆抗体和基于抗体的片段占生物技术市场所有收入的30%以上，用于治疗各种人类疾病，包括哮喘、自身免疫性疾病、细菌和病毒感染、癌症和其他疾病。由于抗体疗法在开发中的药物中占越来越大的比例，随着药物开发成本的不断上升，有效抗体的生产或发现的任何改进都将对人类健康产生重大影响。因此，该提议寻求开发大肠杆菌细胞作为用于快速、低成本表达和分离针对几乎任何感兴趣的靶蛋白抗原的全长人单克隆抗体的技术平台。

项目成果

Efficient expression of full-length antibodies in the cytoplasm of engineered bacteria.

工程细菌细胞质中全长抗体的有效表达。

• DOI: 10.1038/ncomms9072
• 发表时间: 2015-08-27
• 期刊: Nature communications
• 影响因子: 16.6
• 作者: Robinson MP;Ke N;Lobstein J;Peterson C;Szkodny A;Mansell TJ;Tuckey C;Riggs PD;Colussi PA;Noren CJ;Taron CH;DeLisa MP;Berkmen M
• 通讯作者: Berkmen M