Glycosylation of full-length antibodies in Escherichia coli

描述(申请人提供)：目前，治疗性抗体约占所有生物技术收入的30%(&gt；350亿美元)，几乎所有都是在哺乳动物细胞培养中生产的。由于单抗需要糖基化才能提供充分的治疗效果，哺乳动物细胞培养是表达平台的选择。遗憾的是，哺乳动物细胞培养速度慢，成本高，产品异质性大，污染风险大。尽管是增长最快的一类治疗性蛋白质，但对医疗保健消费者来说，单抗的价格可能高得令人望而却步。如果将低复杂性、健壮的细胞用于大规模生产全功能治疗性抗体，生产可能会得到简化。在大肠杆菌中可以产生全长的单抗(免疫球蛋白)，除了结合巨噬细胞Fc受体和诱导效应器功能外，在功能上与哺乳动物细胞中产生的抗体相似。这种不能激发效应器功能的原因是大肠杆菌来源的免疫球蛋白(IGG)的Fc区缺乏糖基化。最近，人们发现空肠弯曲菌天冬酰胺连接(N连接)蛋白的糖基化途径可以功能性地转移到大肠杆菌中，从而赋予蛋白质糖基化的能力。尽管细菌N-葡聚糖在结构上与真核细菌不同，但本研究的目的是确定在糖工程大肠杆菌中的表达是否足以挽救全长免疫球蛋白与效应分子的结合。对这一建议特别感兴趣的是补体依赖的细胞毒途径的C1q受体和抗体依赖的细胞毒途径的Fc3RI和Fc3RIIIa。为了实现这一目标，这项第一阶段SBIR计划的目标是在大肠杆菌中产生第一个能够与效应器结合的全长免疫球蛋白。为了实现这一目标，我们打算(目标1)从糖工程化的大肠杆菌中表达和纯化N-糖基化的免疫球蛋白，并(目标2)确定糖基化是否在体外挽救了与效应分子结合的免疫球蛋白。这些研究具有重要意义，因为它们将(I)探索用于生产治疗性蛋白的范式转换技术平台，(Ii)确定在大肠杆菌中生产糖基化治疗性免疫球蛋白的可行性。 与公共卫生相关：增长最快的一类蛋白质疗法--单抗--不能在大肠杆菌中产生，因为这些简单的细胞天生就不能进行翻译后糖基化。由于大肠杆菌中缺乏糖基化，这些抗体不能引起通常对治疗性抗体疗效至关重要的效应器功能。这些研究的重点是在糖基化能力强的大肠杆菌中产生全长抗体，并确定与效应器分子的结合是否在体外被挽救。

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.