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类非免疫scFv抗体文库中分离抗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，已被许多研究人员用于生成针对无数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进行亲和成熟的scFv抗体工程的理想平台。我们将使用HIV糖蛋白140 （gp140）、gp120-和gp41衍生肽以及完整的病毒粒子对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scFv直接筛选中和HIV。从该文库中分离的nab将进行亲和成熟，以提高中和的广度和效力。亲和成熟的NAb在总体亲和和动力学的开断率上都有所不同，将与亲本NAb进行比较，以中和多种HIV分离物。该提案将提供两个关键问题的见解：1)抗hiv中和性scFv抗体能否从人类scFv抗体非免疫文库中分离出来；2)能否通过提高这些scFv的接通率、切断率和亲和力来改善它们的呼吸和中和效力。这些研究将发现新的抗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.