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 申请中用于开发 HIV 候选疫苗的方法基于 反向疫苗学策略，使用具有广泛和有效作用的人单克隆抗体（mAb） 抗病毒活性，识别病毒包膜 (Env) 上的同源表位，并将这些结构植入 用作免疫原的支架蛋白。然后将这些表位支架免疫原用于体内 引发具有与亲代单克隆抗体相似的抗病毒活性的抗体。 核心 B 的拟议工作分为两个具体目标： 目标 1. 生产项目 1 和 2 中产生的人单克隆抗体。为此，我们将扩大规模 项目 1 和 2 中根据 V2 或 V2/V3 的特异性生产人类单克隆抗体 四级中和表位（QNE）及其介导多种抗病毒功能的能力。我们将 生产足够数量的单克隆抗体以进行广泛的免疫化学、功能和晶体学研究 项目1和2的工作范围要求的研究。这些单克隆抗体将由293个细胞生产 用含有所选单克隆抗体重链和相应轻链基因的质粒转染， 将从抗原特异性 B 细胞中扩增出来。 目标 2. 项目 1、2 和核心 C 的蛋白质生产。为此，我们将生产：a) 生物素标记的 用于对要选择用于 mAb 的抗原特异性 B 细胞进行染色的重组表位支架蛋白 项目 1 和 2 中通过分子方法开发，以及 b) 表位支架蛋白用作 项目 1 和 2 中的抗原以及动物研究核心（核心 C）中的免疫原。这些表位支架 蛋白质将主要在 293 人类胚胎肾细胞或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，这是保存 V2 和 V2/V3 QNE 的构象表位，而大肠杆菌中的生产可能有助于研究 线性表位。 我们在人类抗 HIV-1 单克隆抗体的开发和重组体生产方面拥有丰富的经验 支架蛋白确保了这些试剂的高质量，这对于成功至关重要 完成拟议的 HIVRAD 工作范围。