Monocional Antibody and Protein Core

单克隆抗体和蛋白核心

• 批准号: 8789435
• 负责人: MIROSLAW K GORNY
• 金额: $ 29.47万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-01-23 至 2016-01-22
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8789435
• 关键词: Animals; Antibodies; Antigens; B-Lymphocytes; Biotin; Cells; Complex; Core Protein; Data; Development; Embryo; Ensure; Epitopes; Eukaryotic Cell; Genes; HIV; HIV-1; Human; Human Development; Insecta; Instruction; Light; Mediating; Methods; Molecular; Monoclonal Antibodies; Nature; Oryctolagus cuniculus; Plasmids; Prevention; Production; Proteins; Reagent; Recombinants; Scaffolding Protein; Services; Specificity; Staining method; Stains; Structure; Vaccines; Viral Physiology; Work; base; experience; glycosylation; human monoclonal antibodies; in vivo; kidney cell; monoclonal antibody production; nonhuman primate; scaffold; scale up; three dimensional structure; vaccine candidate; vaccinology; virus envelope

The approach used in this HIVRAD application for the development of HIV candidate vaccines is based on a reverse vaccinology strategy which uses human monoclonal antibodies (mAbs) with broad and potent anti-viral activity to identify cognate epitopes on the virus envelope (Env) and engraft these structures into scaffold proteins to be used as immunogens. These epitope-scaffold immunogens are then used in vivo to elicit Abs with anti-viral activities similar to those displayed by the parental mAbs. The proposed work for Core B is divided into two specific aims: Aim 1. Production of human mAbs generated in Projects 1 and 2. In this aim, we will scale up production of human mAbs generated in Projects 1 and 2 on the basis of their specificity for V2 or for V2/V3 quaternary neutralizing epitopes (QNEs), and their ability to mediate a variety of anti-viral functions. We will produce sufficient quantities of mAbs to perform extensive immunochemical, functional, and crystallographic studies as required by the work scopes of Projects 1 and 2. These mAbs will be produced by 293 cells transfected with plasmids containing the heavy and corresponding light chain genes of selected mAbs which will have been amplified from antigen-specific B cells. Aim 2. Protein production for Project 1, 2 and Core C. In this aim, we will produce: a) biotin-tagged recombinant epitope-scaffold proteins for staining antigen-specific B cells to be selected for mAb development by molecular methods in Projects 1 and 2, and b) epitope-scaffold proteins to be used as antigens in Projects 1 and 2 and as immunogens in Animal Studies Core (Core C). These epitope-scaffold proteins will be produced primarily in 293 human embryonic kidney cells, or alternatively in other mammalian or insect cells. Production in eukaryotic cells will support glycosylation which is needed to preserve the conformational epitopes of V2 and the V2/V3 QNEs, while production in ¿ coli may be useful for the study of linear epitopes. Our extensive experience in the development of human anti-HIV-1 mAbs and production of recombinant scaffolded proteins ensures the high quality of these reagents which are essential for the successful completion ofthe proposed HIVRAD work scope.

HIVRAD应用中用于开发HIV候选疫苗的方法是基于 关于使用人类单克隆抗体（mAb）的反疫苗学策略 抗病毒活性以识别病毒包膜上的同源表位（ENV）并将这些结构植入 脚手架蛋白用于免疫原子。然后将这些表位型损伤免疫在体内使用 与父母mab相似的抗病毒活性引起ABS。 核心B的拟议工作分为两个具体目标： 目标1。项目1和2中产生的人物mab的生产。在此目标中，我们将扩大规模 根据V2或V2/V3的特异性，在项目1和2中产生的人物单元单元生成 第四纪中和表位（QNE）及其介导各种抗病毒功能的能力。我们将 产生足够数量的mAB来执行广泛的免疫化学，功能和晶体学 项目1和2的工作范围的要求。这些mAb将由293个细胞产生 用含有精选mAb的重链轻链基因的质粒转染 将从抗原特异性B细胞中扩增。 目标2。项目1、2和CoreC的蛋白质生产。在此目标中，我们将产生：a）生物素标签 重组表位 - 抗蛋白蛋白用于染色抗原特异性B细胞进行MAB 项目1和2中的分子方法开发，b）用作表位蛋白质蛋白 项目1和2中的抗原以及动物研究核心中的免疫原（核心C）。这些表位损失 蛋白质将主要在293个人类胚胎肾细胞中产生，或者在其他哺乳动物中产生 或昆虫细胞。真核细胞中的生产将支持糖基化，这是维护所需的 V2和V2/V3 QNE的构象表位，而大肠杆菌的产量可能对研究有用 线性表位。 我们在人类抗HIV-1 mAB的发展和重组的生产方面的丰富经验 脚手架蛋白质确保这些试剂的高质量，这对于成功的蛋白质至关重要 拟议的Hivrad工作范围的完成。