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

描述（由申请人提供）：我们将从酵母展示的人非免疫scFv抗体文库中分离抗HIV中和抗体（NAb）。PNNL scFv文库是由太平洋西北国家实验室开发的高多样性文库，已被许多研究人员用于产生针对无数蛋白质抗原的高质量、高亲和力scFv抗体。酵母展示平台对于通过随机诱变的亲和力成熟工程化scFv抗体也是理想的。我们将使用HIV糖蛋白140（gp 140）、gp 120和gp 41衍生肽以及完整病毒粒子对PNNL scFv文库进行选择。我们将介绍一种新的方法来分泌可溶性生物素化的单链抗体，用于直接筛选中和HIV。然后将从该文库分离的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（其总体亲和力以及其动力学结合和解离速率不同）将与其亲本NAb进行比较，以中和多种HIV分离株。该提议将提供对两个关键问题的见解：1）抗HIV中和scFv抗体能否从人scFv抗体的非免疫文库中分离;和2）这些scFv的中和的呼吸和效力能否通过改善它们的结合速率、解离速率和亲和力来改善。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的选择和工程化提供有用的数据。

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.