Antibody Core

摘要 负责抗体工程和生产的核心将服务于项目#4以及核心#1和#3。 将生产和分发多克隆和单克隆抗体。工作流程将被组织起来 沿着4个具体任务的路线。任务1：生产和表征抗聚糖多克隆和单克隆抗体 抗体的免疫后，我们将使用两种主要方法来产生抗体：标准融合， 不朽的合作伙伴，以生产单克隆抗体的“经典”的方式，并表达重组配对 单B细胞抗体链测序后CHO细胞中的重链（H）和轻链（L）（由Project提供 #3）。当需要大量多克隆抗体时，将通过免疫大量的 动物和定期出血。将对单克隆抗体进行亚克隆和同种型分析。纯化后， 感兴趣的抗体将通过表面等离子体共振表征与靶聚糖的结合， Biacore T200。重组伊加和IgM将在昆虫细胞中产生。 任务2：生产抗体Fab片段用于结构研究和其他。结晶是非常重要的 通过去除抗体的Fc片段和产生Fab片段来促进。生产 这些片段的合成将通过两条路线完成。第一个是全长的蛋白水解切割 第二种是瞬时重组表达配对的抗体， CHO细胞中截短的H -全长L链。 任务3：修饰Fc片段以修饰效应子功能。抗菌功能 抗体高度依赖于抗体的Fc片段相关功能。Fc组成 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。 蛋白质如蛋白质A。小鼠和人Fc片段都将被移植和/或突变以修饰Fc片段。 在一些实施方案中，本发明的抗体是具有特定抗体生物活性的抗体，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业务的成功至关重要。 在一个大的合作赠款。所有试剂的电子存档将集中在一台计算机上 并备份在单独的硬盘驱动器以及机构备份系统上。对于每种抗体， 兴趣、免疫原特征、融合日期、同种型、结合常数将在主图中呈现 电子表格H和L序列将可用于从单细胞测序重新表达的抗体。 DNA和细胞将分别储存在-80 ° C和液氮中。完整描述的抗体列表将在下文中列出。 可在PO 1网站上查阅，并定期更新。集团内部的分布将是 在我们的月度会议上讨论。外部分销将遵循MTA协议的机构规则。

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.