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的30％（> 350亿美元），几乎全部在哺乳动物细胞培养中产生。由于单克隆抗体需要糖基化以提供完全治疗的益处，因此哺乳动物细胞培养是选择的表达平台。不幸的是，哺乳动物细胞培养缓慢且昂贵，产物异质性和污染风险很高。尽管生长最快的治疗性蛋白质类别，但单克隆抗体对于医疗保健消费者来说却非常昂贵。如果使用低复杂性，可靠的细胞用于大规模生产功能齐全的治疗抗体，则可以简化生产。全长的单克隆抗体（免疫球蛋白）可以在大肠杆菌中产生，这些抗体在功能上与哺乳动物细胞中产生的抗体相似，除非具有结合巨噬细胞FC受体并引起效应子功能的能力。这种无法引起效应子功能的能力归因于大肠杆菌免疫球蛋白（IgGS）的FC区域缺乏糖基化。最近，发现弯曲杆菌空肠杆菌与天冬酰胺连接（N-连接）蛋白糖基化途径可以在功能上转移到大肠杆菌中，从而赋予糖基化蛋白质的能力。尽管细菌N-聚糖在结构上与其真核对应物不同，但本研究的目的是确定在糖化的大肠杆菌中的表达是否足以挽救全长IgGS与效应分子的结合。该建议特别有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细胞毒性途径的FC3RI和FC3RIIIA。为了实现这一目标，该阶段I SBIR提案的目的是生成能够与效应子结合的大肠杆菌中产生的第一个全长IgG。为了实现这一目标，我们打算（AIM 1）从糖化的大肠杆菌中表达和纯化N-糖基化的IgG，并（AIM 2）确定糖基化是否挽救了IgG在体外与效应分子的结合。这些研究很重要，因为它们将（i）探索用于生产治疗蛋白的范式转移技术平台，并且（ii）确定在大肠杆菌中产生糖基化治疗性IgG的可行性。公共卫生相关性：在大肠杆菌中不能产生最快的蛋白质疗法类别，单克隆抗体，因为这些简单的细胞本质上无力执行转化后糖基化。由于大肠杆菌中缺乏糖基化，这些抗体无法引起效应子功能通常对于治疗抗体的功效至关重要。这些研究的重点是在糖基化竞争力的大肠杆菌中产生全长抗体，并确定在体外挽救与效应分子的结合。