Antibody Core

抗体核心

• 批准号: 10549643
• 负责人: Luc Teyton
• 金额: $ 19.46万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 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: https://reporter.nih.gov/project-details/10549643
• 关键词: Affinity; Agreement; Animals; Anti-Bacterial Agents; Antibodies; Antibody Formation; Antigens; Archives; B-Lymphocytes; Bacterial Infections; Bacterial Proteins; Bacteriology; Binding; Cells; Charge; Chinese Hamster Ovary Cell; Chromatography; Complement; Computers; Crystallization; DNA; Deposition; Development; Dimensions; Electronics; Enzyme-Linked Immunosorbent Assay; Enzymes; Excision; Fab Immunoglobulins; Fc Immunoglobulins; Funding; Grant; Half-Life; Heart; Hemorrhage; Human; Immunization; Immunize; Immunoglobulin A; Immunoglobulin Fragments; Immunoglobulin M; Insecta; Institution; Knowledge; Laboratories; Length; Light; Modification; Monoclonal Antibodies; Mus; Mutate; National Institute of Allergy and Infectious Disease; PTPRC gene; Papain; Polysaccharides; Positioning Attribute; Production; Proteins; Quality Control; Reagent; Recombinant Proteins; Recombinants; Resources; Robot; Role; Route; Running; Surface Plasmon Resonance; System; Techniques; Time; Update; Work; antibody engineering; experience; experimental study; fighting; instrumentation; interest; meetings; member; polyclonal antibody; programs; repository; single cell sequencing; success; web site

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以及核心#1和#3服务。 将生产和分发多克隆和单克隆抗体。工作流程将得到组织。 沿着 4 个具体任务任务 1：生产和表征抗聚糖多克隆和单克隆。 免疫后，我们将使用两种主要方法来生产抗体： 标准融合 以“经典”方式生产单克隆抗体的不朽伙伴，以及重组配对的表达 单 B 细胞抗体链测序后 CHO 细胞中的重 (H) 和轻 (L) 链（由 Project 提供） #3). 当需要大量时，将通过免疫大量来产生多克隆抗体。 动物和常规出血的单克隆抗体将在纯化后进行亚克隆和同种型。 感兴趣的抗体将通过表面等离振子共振来表征与目标聚糖的结合 Biacore T200 将在昆虫细胞中产生。 任务2：生产抗体的Fab片段用于结构研究和其他结晶化是非常重要的。 抗体 Fc 片段的去除和 Fab 片段的产生促进了这一过程。 这些片段的切割将通过两种途径完成。第一种途径是全长的蛋白水解切割。 抗体与木瓜蛋白酶等酶，第二个是配对的瞬时重组表达 CHO 细胞中截短的 H - 全长 L 链。 任务 3：修改 Fc 片段以修改效应器功能。 抗体的 Fc 组成高度依赖于 Fc 片段相关功能。 不仅决定半衰期，还决定补体结合和激活，以及与某些细菌的结合 小鼠和人的 Fc 片段都将被移植和/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、储存和分发 这一特定目标对于核心的成功至关重要。 所有试剂的电子存档将集中在一台计算机上。 并保存在单独的硬盘驱动器以及机构备份系统上。 兴趣、免疫原特征、融合日期、同种型、结合常数将在主文件中呈现 电子表格中的 H 和 L 序列将可用于通过单细胞测序重新表达的抗体。 DNA 和细胞将分别储存在 -80°C 和 LN2 下。完整描述的抗体列表如下。 可在 PO1 网站上获取并定期更新。 我们每月的会议上讨论的外部分配将遵循 MTA 协议的制度规则。