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:
- 金额:$ 8.22万
- 依托单位:
- 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:
- 关键词:AccountingAffectAffinityAffinity ChromatographyAntibodiesAsparagineBindingBiological ProductsCampylobacter jejuniCell Culture TechniquesCell LineCell physiologyCellsCharacteristicsChinese HamsterChinese Hamster Ovary CellComplement 1qComplement-Dependent CytotoxicityComplexCulture MediaDiagnosticDrug KineticsEnzyme-Linked Immunosorbent AssayEscherichia coliFc ReceptorFc domainGelGlycoproteinsGoalsHalf-LifeHandHealthcareHeterogeneityImmuneImmune SeraImmunoglobulin GImmunoglobulinsIn VitroLeadLengthLinkMammalian CellMindModelingMolecular ConformationMonoclonal AntibodiesOrganismOvaryPathway interactionsPharmacologic SubstancePhasePlaguePolysaccharidesPost-Translational Protein ProcessingProcessProductionProductivityProtein GlycosylationProteinsRecombinant ProteinsResearchRiskScreening procedureSmall Business Innovation Research GrantStructureTechnologyTherapeuticTherapeutic Monoclonal AntibodiesTherapeutic antibodiesThermodynamicsTimeTransfectionVariantWestern Blottingantibody-dependent cell cytotoxicitybasecomplement 1q receptorglycosylationimprovedinterestlarge scale productionlink proteinmacrophageprotein expressionpublic health relevancestable cell linesugartherapeutic 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,治疗性抗体占所有生物技术收入的约30%(> 350亿美元),几乎所有抗体都是在哺乳动物细胞培养中产生的。由于单克隆抗体需要糖基化以提供完全的治疗益处,因此哺乳动物细胞培养物是选择的表达平台。不幸的是,哺乳动物细胞培养缓慢且昂贵,具有显著的产物异质性和污染风险。尽管单克隆抗体是增长最快的一类治疗性蛋白质,但对于医疗保健消费者来说可能过于昂贵。如果低复杂性、健壮的细胞用于大规模生产全功能治疗性抗体,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,除了结合巨噬细胞Fc受体和引发效应子功能的能力外,其功能与哺乳动物细胞中产生的抗体相似。这种不能引发效应子功能的现象归因于E.大肠杆菌来源的免疫球蛋白(IgG)。最近发现空肠弯曲菌天冬酰胺连接(N-连接)蛋白糖基化途径可以功能性地转移到大肠杆菌中。大肠杆菌,赋予糖基化蛋白质的能力。虽然细菌N-聚糖在结构上与真核生物的N-聚糖不同,但本研究旨在确定糖工程改造的E。大肠杆菌中的抗体足以拯救全长IgG与效应分子的结合。该提议特别感兴趣的是补体依赖性细胞毒性途径的Clq受体和抗体依赖性细胞毒性途径的Fc 3RI和Fc 3RIIIa。为了实现这一目标,本I期SBIR提案的目标是在大肠杆菌中产生第一个全长IgG。能够与效应子结合的大肠杆菌。为了实现这一点,我们打算(目的1)表达和纯化N-糖基化的IgG从糖工程E。目的2:确定糖基化是否能在体外拯救IgG与效应分子的结合。这些研究是重要的,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和(ii)确定在大肠杆菌中生产糖基化治疗性IgG的可行性。杆菌
公共卫生关系:增长最快的一类蛋白质治疗剂,单克隆抗体,不能在大肠杆菌中生产,因为这些简单的细胞固有地不能进行翻译后糖基化。由于E.在大肠杆菌中,这些抗体不能引发效应子功能,而效应子功能对于治疗性抗体的功效通常是关键的。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。大肠杆菌中,并确定是否结合效应分子在体外获救。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Adam Charles Fisher其他文献
Adam Charles Fisher的其他文献
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{{ truncateString('Adam Charles Fisher', 18)}}的其他基金
Glycoconjugate therapeutic peptides for improved treatment of human diseases
用于改善人类疾病治疗的糖缀合物治疗肽
- 批准号:
8525563 - 财政年份:2013
- 资助金额:
$ 8.22万 - 项目类别:
Production of recombinant human glucocerebrosidase in Escherichia coli
在大肠杆菌中生产重组人葡萄糖脑苷脂酶
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8058360 - 财政年份:2011
- 资助金额:
$ 8.22万 - 项目类别:
Therapeutic antibody fragments from glycoengineered Escherichia coli
来自糖工程大肠杆菌的治疗性抗体片段
- 批准号:
8081020 - 财政年份:2010
- 资助金额:
$ 8.22万 - 项目类别:
Therapeutic antibody fragments from glycoengineered Escherichia coli
来自糖工程大肠杆菌的治疗性抗体片段
- 批准号:
8002633 - 财政年份:2010
- 资助金额:
$ 8.22万 - 项目类别:
Conjugation of polysialic acid to biologics in glycoengineered Escherichia coli
聚唾液酸与糖工程大肠杆菌中的生物制剂结合
- 批准号:
7911940 - 财政年份:2010
- 资助金额:
$ 8.22万 - 项目类别:
Glycophage arrays for the discovery of biomarkers in disease
用于发现疾病生物标志物的噬菌体阵列
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7611816 - 财政年份:2009
- 资助金额:
$ 8.22万 - 项目类别:
Humanizing N-linked glycosylation in Escherichia coli
大肠杆菌中 N 连接糖基化的人源化
- 批准号:
7746389 - 财政年份:2009
- 资助金额:
$ 8.22万 - 项目类别:
Engineering Escherichia coli for glycosylation of complex human proteins
改造大肠杆菌以糖基化复杂的人类蛋白质
- 批准号:
8332786 - 财政年份:2009
- 资助金额:
$ 8.22万 - 项目类别:
Engineering Escherichia coli for glycosylation of complex human proteins
改造大肠杆菌以糖基化复杂的人类蛋白质
- 批准号:
8203830 - 财政年份:2009
- 资助金额:
$ 8.22万 - 项目类别:
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