Isolation of HIV NAbs from a nonimmune yeast-display library of scFv antibodies

从 scFv 抗体非免疫酵母展示文库中分离 HIV NAb

• 批准号: 7418891
• 负责人: Sean Alex Gray
• 金额: $ 28.65万
• 依托单位: SEATTLE BIOMEDICAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-01-01 至 2009-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7418891
• 关键词: Affinity; Antibodies; Antibody Affinity; Antigens; B-Lymphocytes; Bacteriophages; Biological Assay; Data; Diversity Library; Engineering; Enzyme-Linked Immunosorbent Assay; Epitopes; Exhibits; GP 140; Generations; Goals; HIV; HIV Antibodies; HIV Envelope Protein gp120; Human; Human Herpesvirus 4; Immune; Immunoglobulin Fragments; Immunoglobulin G; Kinetics; Laboratories; Libraries; Mediating; Methods; Monoclonal Antibodies; Mus; Mutagenesis; Neutralization Tests; Pacific Northwest; Peptides; Proteins; Rate; Reagent; Research; Research Personnel; Role; Saccharomyces cerevisiae; Screening procedure; Source; Surface; System; Techniques; Testing; Virion; Yeasts; antibody engineering; antigen binding; gp160; improved; insight; neutralizing antibody; novel; vaccine development

DESCRIPTION (provided by applicant): We will isolate anti-HIV neutralizing antibodies (NAbs) from a yeast-displayed library of human nonimmune scFv antibodies. Developed at Pacific Northwest National Laboratory, the PNNL scFv library is a high diversity library which has been used by numerous investigators to generate high-quality, high-affinity scFv antibodies to a myriad of protein antigens. The yeast display platform is also ideal for engineering scFv antibodies by affinity maturation through random mutagenesis. We will perform selections on the PNNL scFv library using HIV glycoprotein 140 (gp140), gp120- and gp41-derived peptides, and intact virions. We will introduce a novel method to secrete soluble biotinylated scFv for direct screening for neutralization of HIV. NAbs isolated from this library will then be subjected to affinity maturation to improve the breadth and potency of neutralization. Affinity matured NAbs, which vary in overall affinity as well as in their kinetic on- and off-rates, will be compared to their parental NAb for neutralization of a diverse panel of HIV isolates. This proposal will provide insights into two key questions: 1) can anti-HIV neutralizing scFv antibodies be isolated from a nonimmune library of human scFv antibodies; and 2) can the breath and potency of neutralization by these scFv be improved upon by improving their on-rates, off-rates, and affinities. These studies will identify new anti-HIV neutralizing antibodies and their epitopes as well as provide useful data for selection and engineering of NAbs isolated from any source.

描述（由申请人提供）：我们将从酵母展示的人类非免疫 scFv 抗体文库中分离出抗 HIV 中和抗体 (NAb)。 PNNL scFv 文库由太平洋西北国家实验室开发，是一个高度多样性的文库，已被众多研究人员用来生成针对多种蛋白质抗原的高质量、高亲和力的 scFv 抗体。酵母展示平台也是通过随机诱变亲和力成熟来工程化 scFv 抗体的理想选择。我们将使用 HIV 糖蛋白 140 (gp140)、gp120 和 gp41 衍生肽以及完整病毒颗粒对 PNNL scFv 文库进行选择。我们将介绍一种分泌可溶性生物素化 scFv 的新方法，用于直接筛选 HIV 中和作用。然后将从该文库中分离出的 NAb 进行亲和力成熟，以提高中和的广度和效力。亲和力成熟的 NAb 在总体亲和力以及动力学结合和解离速率方面有所不同，将与其亲本 NAb 进行比较，以中和不同的 HIV 分离株。该提案将为两个关键问题提供见解：1）能否从非免疫人类 scFv 抗体库中分离出抗 HIV 中和 scFv 抗体？ 2)能否通过提高这些scFv的结合率、解离率和亲和力来提高它们的呼吸和中和效力。这些研究将鉴定新的抗 HIV 中和抗体及其表位，并为从任何来源分离的 Nab 的选择和工程设计提供有用的数据。