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（由申请人提供）：艾滋病毒感染和艾滋病的抗体治疗受病毒避免抗体中和的能力的限制。需要抗体可以在几个不同的进化枝中有效中和HIV-1的临床分离株。该项目的直接目的是通过进一步筛选我们的混合噬菌体 - 播放库（SLE VL -S1-1-VH）来识别和表征催化抗GP120 SCFV，这些库由SLE患者与来自抗GP120抗体（S1-1）的单个VH配对的VL基因组成，从而中上述HIV-1的抗体（S11-1），该抗体（S1-1-1）中上述了HIV-1的几个分离株。由于已显示患有SLE的个体具有能够进行蛋白质催化的光链，因此该目标的假设是，通过GP120的蛋白水解，选择的人类催化光链与人类抗GP120重型链配对，将与人类抗GP120重链配对更有效地中和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动？），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，被动催化抗体在治疗上是有益的，因为常规抗体与单个抗原抗原决定性抗体可逆地结合，但在结合后也可以通过RES从RES中除去循环，而催化抗体可以通过循环抗原抗原抗体（GP120）（cOCLATION）与GP120相结合并灭活。具体目的是（i）识别和序列VL-VH对，将HIV-1 gp120绑定为SCFV-PHAGE和可溶性SCFV（II）测试VL-VH对中和对各种HIV-1分离株的中和对，（III）确定VL-VH对gp120的vl-vh for（kCAT）的定位。实现特定目标的方法包括使用Biotinylated GP120和GP120-CRA筛选SLE VL -SI-SI-I-VH库和“铅” VL-VH对进行催化，使用新鲜PBMC和与蛋白质解相关的蛋白质分析的计算，与与Sudhir Pail博士协作的蛋白质分析的计算，HIV中和中和。在这种新颖的方法中，我们利用一种疾病状态（SLE）为另一种疾病（HIV）开发治疗方法。