Glycosylation of full-length antibodies in Escherichia coli

大肠杆菌中全长抗体的糖基化

基本信息

批准号：
7670053
负责人：
Adam Charles Fisher
金额：
$ 8.22万
依托单位：
GLYCOBIA, INC.
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-15 至 2010-09-14
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7670053
关键词：
Accounting Affect Affinity Affinity Chromatography Antibodies Asparagine Binding Biological Products Campylobacter jejuni Cell Culture Techniques Cell Line Cell physiology Cells Characteristics Chinese Hamster Chinese Hamster Ovary Cell Complement 1q Complement-Dependent Cytotoxicity Complex Culture Media Diagnostic Drug Kinetics Enzyme-Linked Immunosorbent Assay Escherichia coli Fc Receptor Fc domain Gel Glycoproteins Goals Half-Life Hand Healthcare Heterogeneity Immune Immune Sera Immunoglobulin G Immunoglobulins In Vitro Lead Length Link Mammalian Cell Mind Modeling Molecular Conformation Monoclonal Antibodies Organism Ovary Pathway interactions Pharmacologic Substance Phase Plague Polysaccharides Post-Translational Protein Processing Process Production Productivity Protein Glycosylation Proteins Recombinant Proteins Research Risk Screening procedure Small Business Innovation Research Grant Structure Technology Therapeutic Therapeutic Monoclonal Antibodies Therapeutic antibodies Thermodynamics Time Transfection Variant Western Blotting antibody-dependent cell cytotoxicity base complement 1q receptor glycosylation improved interest large scale production link protein macrophage protein expression public health relevance stable cell line sugar therapeutic protein

项目摘要

DESCRIPTION (provided by applicant): Currently, therapeutic antibodies represent approximately 30% of all biotech revenues (>$35 billion) and nearly all are produced in mammalian cell culture. Since monoclonal antibodies require glycosylation to provide full therapeutic benefit, mammalian cell culture is the expression platform of choice. Unfortunately mammalian cell culture is slow and expensive with significant product heterogeneity and contamination risk. Although the fastest growing class of therapeutic proteins, monoclonal antibodies can be prohibitively expensive to the health care consumer. Production could be simplified if low-complexity, robust cells were used for large-scale production of fully functional therapeutic antibodies. Full-length monoclonal antibodies (immunoglobulins) can be produced in Escherichia coli that are functionally similar to those produced in mammalian cells except in the ability to bind macrophage Fc receptors and elicit effector function. This inability to elicit effector function is attributed to the lack of glycosylation in the Fc region of E. coli-derived immunoglobulins (IgGs). Recently, it was discovered that the Campylobacter jejuni asparagine-linked (N-linked) protein glycosylation pathway can be functionally transferred into E. coli, conferring the ability to glycosylate proteins. Although the bacterial N-glycan is structurally distinct from its eukaryotic counterparts, this study aims to determine if expression in glycoengineered E. coli is sufficient to rescue binding of full- length IgGs to effector molecules. Of particular interest to this proposal are the C1q receptor of the complement-dependent cytotoxicity pathway and Fc3RI and Fc3RIIIa of the antibody- dependent cellular cytotoxicity pathway. Towards this goal, the objective of this Phase I SBIR proposal is to generate the first full-length IgGs produced in E. coli capable of binding to effectors. To accomplish this we intend to (Aim 1) express and purify N-glycosylated IgGs from glycoengineered E. coli and (Aim 2) determine if glycosylation rescues IgG binding to effector molecules in vitro. These studies are significant because they will (i) explore a paradigm-shifting technology platform for the production of therapeutic proteins and (ii) determine the feasibility of producing glycosylated therapeutic IgGs in E. coli. PUBLIC HEALTH RELEVANCE: The fastest growing class of protein therapeutics, monoclonal antibodies, cannot be produced in Escherichia coli because these simple cells are inherently incapable of performing post- translational glycosylation. Due to the lack of glycosylation in E. coli, these antibodies are not able to elicit effector functions that are often critical for the efficacy of therapeutic antibodies. The focus of these studies is to produce full-length antibodies in glycosylation-competent E. coli and determine if binding to effector molecules is rescued in vitro.

描述（由申请人提供）：目前，治疗性抗体约占所有生物技术收入（> 350 亿美元）的 30%，并且几乎全部都是在哺乳动物细胞培养物中生产的。由于单克隆抗体需要糖基化才能提供充分的治疗效果，因此哺乳动物细胞培养物是首选的表达平台。不幸的是，哺乳动物细胞培养缓慢且昂贵，并且产品异质性和污染风险显着。尽管单克隆抗体是增长最快的一类治疗蛋白，但对于医疗保健消费者来说可能过于昂贵。如果使用低复杂性、稳健的细胞大规模生产全功能的治疗性抗体，则可以简化生产。全长单克隆抗体（免疫球蛋白）可以在大肠杆菌中产生，其功能与哺乳动物细胞中产生的抗体相似，只是能够结合巨噬细胞 Fc 受体并引发效应功能。这种无法引发效应子功能的原因是大肠杆菌衍生的免疫球蛋白 (IgG) 的 Fc 区缺乏糖基化。最近，人们发现空肠弯曲杆菌天冬酰胺连接（N-连接）蛋白质糖基化途径可以功能性转移到大肠杆菌中，赋予蛋白质糖基化的能力。尽管细菌 N-聚糖在结构上与其真核对应物不同，但本研究旨在确定糖工程大肠杆菌中的表达是否足以挽救全长 IgG 与效应分子的结合。该提案特别感兴趣的是补体依赖性细胞毒性途径的 C1q 受体以及抗体依赖性细胞毒性途径的 Fc3RI 和 Fc3RIIIa。为了实现这一目标，第一阶段 SBIR 提案的目标是生成第一个在大肠杆菌中产生的能够与效应器结合的全长 IgG。为了实现这一目标，我们打算（目标 1）从糖基化大肠杆菌中表达和纯化 N-糖基化 IgG，并（目标 2）确定糖基化是否能在体外挽救 IgG 与效应分子的结合。这些研究意义重大，因为它们将 (i) 探索用于生产治疗性蛋白质的范式转变技术平台，以及 (ii) 确定在大肠杆菌中生产糖基化治疗性 IgG 的可行性。公共健康相关性：增长最快的一类蛋白质治疗药物单克隆抗体无法在大肠杆菌中生产，因为这些简单的细胞本质上无法进行翻译后糖基化。由于大肠杆菌中缺乏糖基化，这些抗体无法引发对于治疗性抗体的功效通常至关重要的效应器功能。这些研究的重点是在具有糖基化能力的大肠杆菌中产生全长抗体，并确定与效应分子的结合是否在体外得到恢复。