Engineered/Proteolytic Antibodies Specific/HIV-1 gp120

工程化/蛋白水解抗体特异性/HIV-1 gp120

• 批准号: 6798857
• 负责人: DOUGLAS F. LAKE
• 金额: $ 22.58万
• 依托单位: ARIZONA STATE UNIVERSITY-TEMPE CAMPUS
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-30 至 2006-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6798857
• 关键词: HIV envelope protein gp120; abzyme; antinuclear autoantibody; genetic screening; immunologic substance development /preparation; neutralizing antibody; peptide library; phage display; protein engineering; systemic lupus erythematosus

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).

描述（申请人提供）：HIV感染和AIDS的抗体治疗受到病毒逃避抗体中和能力的限制。需要能够有效中和几种不同进化枝的HIV-1临床分离株的抗体。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）来鉴定和表征催化性抗gp120 scFv，所述杂交噬菌体展示文库由来自SLE患者的VL基因与来自中和几种HIV-1分离株的抗gp120抗体（S1 - 1）的单个VH配对组成。由于SLE患者已显示具有能够蛋白质催化的轻链，因此该目标的假设是，通过gp120的蛋白水解，与人抗gp120重链配对的所选人催化轻链将比亲本IgG更有效地中和HIV。基础科学长期目标：了解蛋白水解机制（即丝氨酸蛋白酶样活性？）用于选定的催化抗体，并提高其周转率，同时保持特异性;临床长期目标：与常规抗体相比，催化抗体的被动施用在治疗上是有益的，因为常规抗体可逆地结合单个抗原决定簇，但也可以在结合后通过RES从循环中除去，而催化性抗体可以在从循环中去除之前通过切割靶抗原（GP 120）结合并降解。具体目的是（i）鉴定和测序结合和切割HIV-1 gp120作为scFv-噬菌体和可溶性scFv的VL-VH对，（ii）测试VL-VH对中和不同的HIV-1分离物，和（iii）确定VL-VH对gp120底物的转换速率（kcat），以定位我们用于产生临床有用的IgG。实现具体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"前导" VL-VH对的催化作用，使用新鲜PBMC用不同临床分离株进行HIV中和测定，以及与Sudhir Paul博士合作计算蛋白水解速率。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。