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Developing a screening campaign for immune enhancers

开展免疫增强剂筛选活动

基本信息

批准号：
9528448
负责人：
ADAM ZWEIFACH
金额：
$ 30.96万
依托单位：
UNIVERSITY OF CONNECTICUT STORRS
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-08-01 至 2021-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9528448
关键词：
Accidents Affect Agonist Antibodies Antigen Presentation Attention B-Lymphocytes Bar Codes Binding Biological Assay CD3 Antigens CD80 Antigens Cells Cellular biology Collection Cytoplasmic Granules Cytotoxic T-Lymphocytes Data Dendritic Cells Development Dimethyl Sulfoxide End Point Assay Enhancers Exocytosis Flow Cytometry Fluorescent Antibody Technique Future Goals Helper-Inducer T-Lymphocyte Hour Human Imiquimod Immune Immune response Immunity Immunological Models Institutes LAMP-1 Label Length Libraries Lytic Measures Methods Molecular Bank Monitor Monoclonal Antibodies Monoclonal Antibody HuM291 Phenotype Population Production Recombinant Cytokines Source T-Cell Receptor Testing Thalidomide Therapeutic Time Toll-like receptors United States National Institutes of Health Validation analog antiviral immunity base cancer therapy cell killing cell type cellular targeting cytokine design experimental study follow-up high throughput screening immune function innovation interest lenalidomide miniaturize novel novel therapeutics repository response screening small molecule small molecule libraries success therapy design vaccine efficacy

项目摘要

Therapies designed to enhance immunity are becoming important components of cancer therapies, and could be used to increase anti-viral immunity and augment the efficacy of vaccines. Many strategies focus on biologic agents such as recombinant cytokines or monoclonal antibodies. However, the success of imiquimod and lenalidomide, the two available small molecule immune-enhancers, suggests that additional agents of this type could be useful. One reason that there are so few small-molecule immune enhancers is that there has historically been no way to conduct high-throughput screening (HTS) to find them. Imiquimod and thalidomide analogs were found to enhance immune responses by accident. We have developed an assay that should finally make it possible to screen large compound libraries for immune-enhancing small molecules. The assay is a powerful revision of an HTS that we applied successfully to the NIH's Molecular Libraries Small Molecule Repository. In the new enhanced assay, we stimulate TALL- 104 human leukemic cytotoxic T lymphocytes (CTLs) with beads coated with anti-CD3 antibodies, generating submaximal exocytosis specifically in the bead-bound population. We monitor exocytosis by measuring binding of a fluorescently-labeled antibody against LAMP-1 (CD107a) to cells using flow cytometry. This allows us to conduct a no-wash assay that can detect compounds that enhance of exocytosis and discriminate them from compounds that cause exocytosis on their own. TALL-104 CTLs serve in our strategy as both a model of an immunologically-relevant cell type as well as a surrogate for other immune cell types for which it would be difficult to devise HTS-ready assays. Conducting a screen with TALL-104 cells followed by assessment of the effects of hits on other important immune functions will likely lead to the identification of therapeutic leads or to probes that could be used to identify novel cellular targets that can be exploited to produce immune enhancement. Our three aims are designed are to 1) develop and optimize a highly sophisticated assay that will assess the effects of treating cells with compounds for different lengths of time in a single read step, then validate the optimized assay by screening the Prestwick Compound Library; 2) develop a set of secondary tests to examine the effect of hits on target cell killing by CTLs and representative functions of helper T cells, B cells and dendritic cells and 3) validate the HTS/ follow-up strategy by screening the Broad Institute's DOS informer collection of ~10K compounds. Completing our aims will provide an assay and follow-up workflow suitable for screening larger compound collections, as well as generating a number of candidate molecules obtained from screening the Prestwick and Broad collections that can be pursued in future experiments.

旨在增强免疫力的疗法正在成为癌症治疗的重要组成部分，并可能用于提高抗病毒免疫力，增强疫苗效力。许多战略都侧重于生物制剂，如重组细胞因子或单抗。然而，咪喹莫特的成功而来那度胺，这两种可用的小分子免疫增强剂，表明更多的这种药物类型可能会很有用。小分子免疫增强剂如此之少的一个原因是从历史上看，无法进行高通量筛查(HTS)来找到它们。咪喹莫特和沙利度胺类似物被意外地发现可以增强免疫反应。我们已经开发了一种分析方法，最终应该可以筛选大型化合物文库增强免疫力的小分子。这项测试是我们成功应用的HTS的强大修订版到美国国立卫生研究院的分子图书馆小分子资料库。在新的增强化验中，我们刺激高个子- 104个人白血病细胞毒性T淋巴细胞(CTL)，用包被抗CD3抗体的珠子，产生次极大的胞吐作用，特别是在结合珠粒的人群中。我们通过测量结合来监测胞吐作用。用流式细胞术将抗LAMP-1(CD107a)的荧光标记抗体应用于细胞。这使我们能够进行免清洗检测，可以检测到增强胞吐作用的化合物，并将它们与会自行引起胞吐的化合物。Tall-104 CTL在我们的战略中既是免疫相关的细胞类型以及它可能是的其他免疫细胞类型的替代品很难设计出适合HTS的检测方法。用TALL-104细胞进行筛查，然后评估 HITS对其他重要免疫功能的影响可能会导致确定治疗线索或可用于识别可用于产生免疫的新细胞靶点的探针增强功能。我们的三个目标是：1)开发和优化一种高度复杂的检测方法，以评估在一次读取步骤中用不同时间的化合物处理细胞的效果，然后验证通过筛选Prestwick化合物文库来优化检测方法；2)开发一套二次检测来检验 HITS对CTL杀伤靶细胞及辅助性T细胞、B细胞和CD8~+细胞代表功能的影响树突状细胞和3)通过筛选远大研究所的DOS告密者来验证HTS/随访策略收集~10K个化合物。完成我们的目标将提供适合以下方面的测试和后续工作流程筛选更大的化合物集合，以及生成从以下来源获得的许多候选分子筛选Prestwick和BRoad的收藏品，以便在未来的实验中继续进行。