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: 7294568
• 负责人: DOUGLAS F. LAKE
• 金额: $ 22.42万
• 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 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7294568
• 关键词: HIV envelope protein gp120; abzyme; antinuclear autoantibody; genetic screening; immunologic substance development /preparation; neutralizing antibody; peptide library; 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（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的能力的限制。需要能够有效中和多个不同进化枝中的 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混合噬菌体展示文库 (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 scFv 的单个 VH 配对组成。 -gp120 抗体 (S1-1)，可中和多种 HIV-1 分离株。由于 SLE 患者已被证明拥有能够进行蛋白质催化的轻链，因此该目标的假设是，与人抗 gp120 重链配对的选定人催化轻链将比亲本 IgG 更有效地中和 HIV，通过水解GP120。基础科学长期目标：了解所选催化抗体的蛋白水解机制（即丝氨酸蛋白酶样活性？），并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，被动施用催化抗体在治疗上有益，因为传统抗体可逆地结合单个抗原决定簇，但在结合后也可能被 RES 从循环中去除，而催化抗体可以结合和在从循环中去除之前通过切割靶抗原 (gp120) 来灭活。具体目标是 (i) 鉴定并测序结合并切割 HIV-1 gp120 作为 scFv 噬菌体和可溶性 scFv 的 VL-VH 对，(ii) 测试 VL-VH 对对不同 HIV-1 分离株的中和作用，以及 (iii) ) 确定 gp120 底物的 VL-VH 对的周转率 (kcat)，以帮助我们生成临床上有用的 IgG。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“引导”VL-VH 对进行催化，使用新鲜 PBMC 对不同临床分离株进行 HIV 中和测定，以及计算与 Sudhir Paul 博士合作研究蛋白水解率。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。