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

描述(申请人提供)：我们将从酵母展示的人类非免疫性单链抗体文库中分离出抗HIV中和抗体(NAB)。PNNL单链抗体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来产生针对多种蛋白质抗原的高质量、高亲和力的单链抗体。酵母展示平台也是通过随机突变亲和力成熟来设计单链抗体的理想平台。我们将使用HIV糖蛋白140(Gp140)、gp120和gp41衍生的多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分泌可溶性生物素化单链抗体的方法，用于直接筛选HIV中和抗体。从该文库中分离的NAB将经过亲和成熟，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态开启和关闭速率方面存在差异，将与它们的亲本NAB进行比较，以中和一组不同的艾滋病毒分离株。这项提议将提供对两个关键问题的见解：1)抗HIV中和单链抗体能否从非免疫性人源ScFv抗体库中分离出来；2)这些单链抗体能否通过改善它们的开通率、OFF率和亲和力来改善其中和呼吸和效力。这些研究将确定新的抗艾滋病毒中和抗体及其表位，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.