Engineered/Proteolytic Antibodies Specific/HIV-1 gp120

描述（由申请人提供）：HIV感染和艾滋病的抗体治疗受到病毒逃避抗体中和能力的限制。需要一种抗体，能够有效地中和临床分离的HIV-1在几个不同的分支。该项目的直接目标是通过进一步筛选我们的杂交噬菌体展示文库（SLE VL -S1-1-VH）来鉴定和表征催化抗gp120 scFv，该文库由来自SLE患者的VL基因与来自抗gp120抗体（S1-1）的单个VH配对组成，该抗体可以中和几种HIV-1分离株。由于SLE患者已被证明具有能够催化蛋白质的轻链，因此该目标的假设是，选择的人催化轻链与人抗gp120重链配对，通过gp120的蛋白水解，比亲本IgG更有效地中和HIV。基础科学长期目标：了解选定的催化抗体的蛋白水解机制（即丝氨酸蛋白酶样活性？），在保持特异性的同时提高其周转率；临床长期目标：与传统抗体相比，被动给药催化抗体在治疗上是有益的，因为传统抗体可逆地与单一抗原决定因子结合，但也可能在结合后被RES从循环中去除，而催化抗体在从循环中去除之前可以通过切割靶抗原（gp120）结合并灭活。具体目的是：(i)鉴定和测序结合和切割HIV-1 gp120的VL-VH对，作为scFv噬菌体和可溶性scFv； （ii）测试VL-VH对对中和各种HIV-1分离物的作用；（iii）确定gp120底物的VL-VH对的周转率（kcat），以便为产生临床有用的IgG定位。实现特定目标的方法包括使用生物素化的gp120和gp120- cra筛选SLE VL -SI-I-VH文库和“先导”VL- vh对进行催化，使用新鲜的PBMC对不同临床分离株进行HIV中和试验，以及与Sudhir Paul博士合作计算蛋白质水解率。在这种新方法中，我们利用一种疾病状态（SLE）来开发另一种疾病状态（HIV）的治疗方法。

DESCRIPTION (PROVIDED BY APPLICANT): Antibody therapy of HIV infection and AIDS has been limited by the ability of the virus to escape antibody neutralization. There is a need for antibodies that can potently neutralize clinical isolates of HIV-1 across several diverse clades. The immediate objective of this project is to identify and characterize catalytic anti-gp120 scFv by further screening our hybrid phage-display library (SLE VL -S1-1-VH ) consisting of VL genes from SLE patients paired with a single VH from an anti-gp120 antibody (S1-1) that neutralizes several isolates of HIV-1. Since individuals with SLE have been shown to possess light chains capable of protein catalysis, the hypothesis for this objective is that selected human catalytic light chains paired with a human anti-gp120 heavy chain will more effectively neutralize HIV than the parent IgG, by proteolysis of gp120. Basic science long-term objectives: Understand the proteolytic mechanism (i.e. serine protease-like activity?) for selected catalytic antibodies and improve their turnover rates while maintaining specificity; Clinical long-term objectives: Passive administration of a catalytic antibody would be therapeutically beneficial compared to conventional antibodies because conventional antibodies bind reversibly to a single antigenic determinant, but may also be removed from circulation by the RES after binding, while catalytic antibodies can bind and inactivate by cleaving target antigen (gp120) prior to removal from circulation. Specific Aims are to (i) identify and sequence VL-VH pairs that bind and cleave HIV-1 gp120 as scFv-phage and soluble scFv (ii) test the VL-VH pair for neutralization of diverse HIV-1 isolates, and (iii) determine the turnover rate (kcat) of the VL-VH pair for gp120 substrate to position us for generation of a clinically useful IgG. Methods to achieve the specific aims include screening the SLE VL -SI-I-VH library and "lead" VL-VH pairs for catalysis using biotinylated gp120 and gp120-CRA, HIV neutralization assays with diverse clinical isolates using fresh PBMC, and calculation of the rate of proteolysis in collaboration with Dr. Sudhir Paul. In this novel approach we take advantage of one disease state (SLE) to develop a treatment for another (HIV).