Antibody Core
抗体核心
基本信息
- 批准号:10549643
- 负责人:
- 金额:$ 19.46万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2023
- 资助国家:美国
- 起止时间:2023-05-09 至 2028-04-30
- 项目状态:未结题
- 来源:
- 关键词:AffinityAgreementAnimalsAnti-Bacterial AgentsAntibodiesAntibody FormationAntigensArchivesB-LymphocytesBacterial InfectionsBacterial ProteinsBacteriologyBindingCellsChargeChinese Hamster Ovary CellChromatographyComplementComputersCrystallizationDNADepositionDevelopmentDimensionsElectronicsEnzyme-Linked Immunosorbent AssayEnzymesExcisionFab ImmunoglobulinsFc ImmunoglobulinsFundingGrantHalf-LifeHeartHemorrhageHumanImmunizationImmunizeImmunoglobulin AImmunoglobulin FragmentsImmunoglobulin MInsectaInstitutionKnowledgeLaboratoriesLengthLightModificationMonoclonal AntibodiesMusMutateNational Institute of Allergy and Infectious DiseasePTPRC genePapainPolysaccharidesPositioning AttributeProductionProteinsQuality ControlReagentRecombinant ProteinsRecombinantsResourcesRobotRoleRouteRunningSurface Plasmon ResonanceSystemTechniquesTimeUpdateWorkantibody engineeringexperienceexperimental studyfightinginstrumentationinterestmeetingsmemberpolyclonal antibodyprogramsrepositorysingle cell sequencingsuccessweb site
项目摘要
Abstract
The core in charge of antibody engineering and production will serve projects #4, as well as Cores #1 and #3.
Polyclonal as well as monoclonal antibodies will be produced and distributed. The flow of work will be organized
along the lines of 4 specific tasks. Task 1: Produce and characterize anti-glycan polyclonal and monoclonal
antibodies. Following immunization, we will use two main approaches to produce antibodies: standard fusion to
an immortal partner to produce monoclonal antibodies the “classical” way, and expression of recombinant paired
Heavy (H) and Light (L) chains in CHO cells after single B cell antibody chain sequencing (provided by Project
#3). Polyclonal antibodies when needed in large quantities will be produced by immunizing large number of
animals and regular bleeding. Monoclonal antibodies will be subcloned and isotyped. After purification,
antibodies of interest will be characterized for binding to the target glycan by surface plasmon resonance on a
Biacore T200. Recombinant IgA and IgM will be produced in insect cells.
Task 2: Produce Fab fragments of antibodies for structural studies and others. Crystallization is greatly
facilitated by the removal of the Fc fragment of antibodies and the production of Fab fragments. The production
of these fragments will be accomplished by two routes. The first one is the proteolytic cleavage of the full-length
antibodies with enzymes such as papain, the second one is the transient recombinant expression of paired
truncated H - full length L chains in CHO cells.
Task 3: Modifications of the Fc fragment to modify effector functions. The antibacterial functions of
antibodies are highly dependent on the Fc fragment-associated functions of antibodies. Fc composition
determines not only half-life but also complement binding and activation, as well as binding to some bacterial
protein such as protein A. Both mouse and human Fc fragments will be grafted and/or mutated to modify the
bioactivity of a particular antibody and expressed in a CHO cell recombinant expression system.
Task 4: Quality control, storage, and distribution. This particular aim is essential to the success of a core
within a large collaborative grant. Electronic archiving of all reagents will be centralized on a single computer
and backed up on a separate hard drive as well as on the institutional backup system. For each antibody of
interest, immunogen characterization, date of fusion, isotype, binding constants will be presented in a master
spreadsheet. H and L sequences will be accessible for antibodies re-expressed from single cell sequencing.
DNA and cells will be stored at -80ºC and LN2, respectively. The list of the fully described antibodies will be
available on the Website of the PO1 and updated on a regular basis. Distribution within the group will be
discussed at our monthly meetings. Outside distribution will follow the institutional rules of MTA agreement.
抽象的
负责抗体工程和生产的核心将为项目#4提供服务,以及核心#1和#3。
将产生和分布多克隆和单克隆抗体。工作流将组织
沿着4个特定任务的行。任务1:生产和表征抗聚糖多克隆和单克隆
抗体。免疫后,我们将使用两种主要方法来产生抗体:标准融合到
不朽的伴侣产生单克隆抗体的“经典”方式,并表达重组配对
单个B细胞抗体链测序后CHO细胞中的重(H)和光(L)链(由Project提供
#3)。多克隆抗体在需要时会通过大量免疫数量产生
动物和常规出血。单克隆抗体将被亚克隆和同型。纯化后,
感兴趣的抗体将通过表面等离子体共振在A上与靶向聚糖结合来表征
Biacore T200。重组IgA和IgM将在昆虫细胞中产生。
任务2:生成用于结构研究和其他抗体的晶圆厂片段。结晶很棒
通过去除抗体的FC片段和Fab片段的产生来促进。生产
这些碎片将通过两条路线来完成。第一个是全长的蛋白水解切割
带有帕帕因(Papain)酶的抗体,第二种是配对的瞬态重组表达
CHO细胞中截短的H-全长L链。
任务3:修改FC片段以修改效应子功能。抗菌功能
抗体高度依赖于Fc片段相关的抗体功能。 FC组成
不仅确定半衰期,还可以补充结合和激活,并与某些细菌结合
蛋白质等蛋白质A。小鼠和人FC片段都将被分级和/或突变以修饰
特定抗体的生物活性,并在CHO细胞重组表达系统中表达。
任务4:质量控制,存储和分配。这个特殊目标对于核心的成功至关重要
在大型合作赠款中。所有试剂的电子存档将集中在一台计算机上
并在单独的硬盘驱动器以及机构备份系统上备份。对于每种抗体
兴趣,免疫原性,融合日期,同种型,结合常数将在主机中呈现
电子表格。对于从单细胞测序重新表达的抗体,H和L序列将可访问。
DNA和细胞将分别存储在-80ºC和LN2下。完全描述的抗体的列表将是
在PO1网站上可用,并定期更新。小组内的分布将是
在我们的每月会议上讨论。外部分配将遵循MTA协议的机构规则。
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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Luc Teyton其他文献
Luc Teyton的其他文献
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{{ truncateString('Luc Teyton', 18)}}的其他基金
Molecular basis of glycan recognition by T and B cells
T 和 B 细胞识别聚糖的分子基础
- 批准号:
10549648 - 财政年份:2023
- 资助金额:
$ 19.46万 - 项目类别:
Leveraging Human iPSC-derived beta-cells to Probe Antigen Specificity of Anti-islet Memory T Cells in T1D
利用人 iPSC 衍生的 β 细胞探测 T1D 中抗胰岛记忆 T 细胞的抗原特异性
- 批准号:
10589556 - 财政年份:2023
- 资助金额:
$ 19.46万 - 项目类别:
Multidimensional development of high-affinity anti-glycan antibodies to fight deadly bacterial infections
多维开发高亲和力抗聚糖抗体以对抗致命细菌感染
- 批准号:
10549640 - 财政年份:2023
- 资助金额:
$ 19.46万 - 项目类别:
Mechanistic Studies of Combination Adjuvants to Target B Cells in Vaccines
疫苗中针对 B 细胞的组合佐剂的机理研究
- 批准号:
10599324 - 财政年份:2021
- 资助金额:
$ 19.46万 - 项目类别:
Mechanistic Studies of Combination Adjuvants to Target B Cells in Vaccines
疫苗中针对 B 细胞的组合佐剂的机理研究
- 批准号:
10218993 - 财政年份:2021
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Mechanistic Studies of Combination Adjuvants to Target B Cells in Vaccines
疫苗中针对 B 细胞的组合佐剂的机理研究
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10397167 - 财政年份:2021
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Early diagnosis and mechanistic studies of type 1 diabetes using single cell analysis
使用单细胞分析进行 1 型糖尿病的早期诊断和机制研究
- 批准号:
10362605 - 财政年份:2019
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Early diagnosis and mechanistic studies of type 1 diabetes using single cell analysis
使用单细胞分析进行 1 型糖尿病的早期诊断和机制研究
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9884757 - 财政年份:2019
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Increasing the Social Science Impact of Biomedical Research
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10089675 - 财政年份:2018
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$ 19.46万 - 项目类别:
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