NOVEL MONOCLONAL ANTIBODIES FOR INVESTIGATING GP120

用于研究 GP120 的新型单克隆抗体

基本信息

批准号：
2766682
负责人：
Thomas G Evans
金额：
$ 23.85万
依托单位：
UNIVERSITY OF ROCHESTER
依托单位国家：
美国
项目类别：
财政年份：
1998
资助国家：
美国
起止时间：
1998-09-30 至 2000-09-29
项目状态：
已结题

项目摘要

DESCRIPTION (Adapted from the applicant's abstract): Infection by HIV-1 continues to be a leading cause of death among children and adults throughout the world. Despite recent advances in anti-retroviral therapy, ultimate control of infection will depend upon development of effective vaccines. However, currently available HIV-1 vaccines have failed to elicit broadly reactive antibodies that neutralize significant numbers of primary isolates. In addition, monoclonal antibodies (mAbs) made to date rarely neutralize primary isolates of HIV-1, and the few which do broadly neutralize have little effect on the primary isolate utilization of two of the key HIV-1 co-receptors, either CXCR4 or CCR5. However, sera from highly selected long-term non-progressors broadly neutralize primary isolates more effectively than any available mAb. The applicants hypothesize that these sera contain antibodies that can bind to the conformational determinant(s) on the CD4-gp120 complex that HIV-1 utilizes to bind to the CCR5 co-receptor. The investigators propose that these specific epitopes can be best defined both structurally and genetically by cloning the cells producing mAbs from individuals whose sera show such broad neutralization capability. Since clinical data reveals that this co-receptor is essentially required for infection with HIV-1, further definition of this binding site will be critical in the rational design of an effective HIV-1 immunogen. To test the above hypothesis, sera will be screened from HIV-1 long-term non-progressors (LTNP) for neutralization of R5 isolates in a standard peripheral blood mononuclear cell (PBMC) assay. These sera also will be tested for the ability to block the infection of CD4/CCR5-expressing cell lines by R5 isolates. The three patients whose sera have the highest titers of such activity will be selected for bone marrow biopsy, and a single phage display antibody library (PDL) will be constructed. The PDL will be sequentially panned against multiple R5 gp120/CD4 complexes to positively select for broadly neutralizing antibodies. Negative panning strategies against T cell line tropic gp120 and linear V3 determinants will be employed to remove binding antibodies of lesser interest. Antibodies produced by 100 selected clones then will be screened using neutralization assays and CCR5 binding assays. The best 25 antibodies will be defined carefully for their breadth and degree of neutralization and their ability to be mutated to produce reagents with greater neutralizing capability. These antibodies will be used to define structural, biologic, and genetic features of R5 primary isolates that are critical in the envelope-CCR5 interaction and in the development of an effective immunogen.

描述（改编自申请人的摘要）：HIV-1 感染仍然是儿童和成人死亡的主要原因全世界。尽管抗逆转录病毒疗法最近取得了进展，感染的最终控制将取决于有效的开发疫苗。然而，目前可用的 HIV-1 疫苗未能引起具有广泛反应性的抗体，可以中和大量的初级抗体隔离。此外，迄今为止制造的单克隆抗体（mAb）很少中和 HIV-1 的主要分离株，以及少数具有广泛作用的分离株中和对其中两种的主要分离株利用影响不大关键的 HIV-1 辅助受体，CXCR4 或 CCR5。然而，血清来自高度选定的长期非进展者更广泛地中和初级分离株比任何可用的单克隆抗体都有效。申请人假设这些血清含有可以结合构象决定簇的抗体 HIV-1 利用 CD4-gp120 复合物与 CCR5 结合共受体。研究人员提出，这些特定表位可以通过克隆细胞在结构和遗传上得到最好的定义从血清显示出如此广泛中和作用的个体中生产单克隆抗体能力。由于临床数据表明该共同受体是感染 HIV-1 所必需的，对此的进一步定义结合位点对于合理设计有效的 HIV-1 至关重要免疫原。为了检验上述假设，将长期筛查血清中的 HIV-1 用于中和标准中 R5 分离株的非进展菌 (LTNP) 外周血单核细胞（PBMC）测定。这些血清也将测试阻断 CD4/CCR5 表达细胞感染的能力 R5 分离株。血清滴度最高的三名患者具有这种活性的噬菌体将被选择用于骨髓活检，并且单个噬菌体将构建展示抗体库（PDL）。 PDL 将是依次对多个 R5 gp120/CD4 复合物进行淘选，以得到阳性结果选择广泛中和的抗体。负面淘选策略将采用针对 T 细胞系 tropic gp120 和线性 V3 决定簇去除不太感兴趣的结合抗体。 100 产生的抗体然后将使用中和测定和 CCR5 筛选选定的克隆结合测定。最好的 25 种抗体将根据其特性仔细定义中和的广度和程度及其突变的能力生产具有更强中和能力的试剂。这些抗体将用于定义 R5 的结构、生物学和遗传特征在包膜-CCR5 相互作用和开发有效的免疫原。