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
基本信息
- 批准号:8200628
- 负责人:
- 金额:$ 21.34万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 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:
- 关键词:AccountingAffinityAntibodiesAntibody TherapyAntigen TargetingAntigensAsthmaAutoimmune DiseasesAvidityB-LymphocytesBackBacterial InfectionsBasic ScienceBindingBinding ProteinsBiochemical ProcessBiological AssayBiological MarkersBiotechnologyBypassCapitalCell Surface ProteinsCell surfaceCellsChromosomesCytoplasmCytoplasmic ProteinCytosineDevelopmentDiagnosisDiagnosticDiseaseEndotheliumEngineeringEscherichia coliExhibitsFc domainFermentationFutureGenesGenetic EngineeringGoalsGrowthHalf-LifeHealthHumanHybridomasImmunoglobulin FragmentsImmunoglobulin GImmunoglobulinsKidneyLengthLibrariesLifeLinkMalignant NeoplasmsMammalian CellMammalsMarketingMethodsMonoclonal AntibodiesNoisePathway interactionsPhage DisplayPharmaceutical PreparationsPlasmidsProcessProductionPropertyProtein FragmentProtein SubunitsProteinsProteolysisRecombinantsReporterReportingRibosomal RNASalesScreening procedureSerumSignal TransductionSpecificityStructureSystemTechnologyTertiary Protein StructureTestingTherapeuticTherapeutic AgentsTimeTransgenic MiceTreatment EfficacyViralVirus Diseasesantigen bindingassay developmentbasecancer cellcellular engineeringcombinatorialcostdesigndisulfide bonddrug developmentextracellularglycosylationhuman diseasehuman monoclonal antibodiesinorganic phosphateinterestmicroorganismmolecular sizenovelperiplasmpreventpromoterprotein expressionprotein protein interactionrapid growththerapeutic developmenttherapeutic proteintoolyeast protein
项目摘要
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) 在人类健康方面有着巨大的前景,其应用范围从针对癌细胞的治疗剂到可以检测痕量水平的给定抗原的诊断生物标志物。这一承诺在2007年全球抗体销售额达到近310亿美元中得到了最好的体现,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对几乎任何目标抗原具有高亲和力和特异性的人类单克隆抗体和抗体衍生片段。从治疗的角度来看,全长 mAb 或 IgG 通常比较小的抗体片段更有优势,因为它们在哺乳动物中的循环半衰期较长,这是由于它们的大分子尺寸阻止了肾脏的清除,并且能够避免通过使用补救途径在内皮中进行蛋白水解。然而,由于这些多亚基蛋白质的复杂性,它们的生产很大程度上限于真核表达系统,例如CHO或杂交瘤细胞,因此是麻烦的、昂贵的、耗时的并且不适合并行化。由于这些缺点,现有的 IgG 发现和生产技术难以跟上对这些重要生物分子快速增长的需求。为了帮助缩小与 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、易于使用和低成本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(例如 Fvs、scFvs、Fab 或 F(ab')2s)的极佳宿主,但其 IgG 表达和工程化的潜力尚未得到彻底研究。因此,该提案的目标是将大肠杆菌开发为发现、工程和制造全长人类 IgG 的强大工具。在具体目标 1 下,将设计一种新型大肠杆菌菌株,专门用于在细胞质区室中高水平表达重组 IgG。该提案的具体目标 2 旨在开发一种独特的筛选方法,用于直接选择“细胞克隆”——从活大肠杆菌细胞质中分离出来的针对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靶蛋白抗原的细胞克隆的大型 IgG 序列合成文库来证明。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 的工具包,这些 IgG 可用于基础研究、诊断和治疗。
公共健康相关性: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,用于治疗多种人类疾病,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。由于抗体疗法在正在开发的药物中所占的比例越来越大,并且随着药物开发成本的不断增加,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,该提案旨在开发大肠杆菌细胞作为技术平台,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。
项目成果
期刊论文数量(1)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
Efficient expression of full-length antibodies in the cytoplasm of engineered bacteria.
全长抗体在工程细菌细胞质中的高效表达。
- DOI:
- 发表时间:2015-08-27
- 期刊:
- 影响因子:16.6
- 作者:Robinson, Michael;Ke, Na;Lobstein, Julie;Peterson, Cristen;Szkodny, Alana;Mansell, Thomas J;Tuckey, Corinna;Riggs, Paul D;Colussi, Paul A;Noren, Christopher J;Taron, Christopher H;DeLisa, Matthew P;Berkmen, Mehmet
- 通讯作者:Berkmen, Mehmet
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Mehmet Berkmen其他文献
Mehmet Berkmen的其他文献
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