Developing a screening campaign for immune enhancers

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， 用于提高抗病毒免疫力和增强疫苗的效力。许多战略都侧重于 生物制剂如重组细胞因子或单克隆抗体。然而，咪喹莫特的成功 和来那度胺，这两种可用的小分子免疫增强剂，表明这种额外的药物 类型可能有用。小分子免疫增强剂如此之少的一个原因是 历史上没有办法进行高通量筛选（HTS）来找到它们。咪喹莫特和沙利度胺 意外发现类似物可以增强免疫反应。 我们已经开发了一种检测方法，最终可以筛选大型化合物库， 免疫增强小分子。该分析是一个强有力的修订高温超导，我们成功地应用 美国国立卫生研究院的分子图书馆小分子储存库。在新的增强试验中，我们刺激TALL- 104个人白血病细胞毒性T淋巴细胞（CTL）与抗CD 3抗体包被的珠粒，产生 特别是在珠结合群体中的次最大胞吐作用。我们通过测量结合来监测胞吐作用 使用流式细胞术将荧光标记的抗LAMP-1（CD 107 a）的抗体与细胞接触。这使我们能够 进行无洗涤试验，该试验可以检测增强胞吐作用的化合物，并将其与 能引起胞吐作用的化合物在我们的策略中，TALL-104 CTL既作为一个模型， 免疫相关细胞类型以及其将被用于的其它免疫细胞类型的替代物 很难设计出HTS就绪的测定法。用TALL-104细胞进行筛选，然后评估TALL-104细胞的细胞毒性。 命中对其他重要免疫功能的影响将可能导致治疗线索的鉴定， 可用于识别新的细胞靶点的探针， 增强 我们的三个目标是：1）开发和优化一种高度复杂的测定方法， 在单个读取步骤中用化合物处理细胞不同时间长度的效果，然后验证 通过筛选Prestwick化合物文库优化测定; 2）开发一组二级测试以检查 通过CTL和辅助T细胞、B细胞和辅助T细胞的代表性功能， 树突状细胞和3）通过筛选布罗德研究所的DOS告密者来验证HTS/随访策略 约10 K化合物的集合。完成我们的目标将提供一个适合的分析和后续工作流程， 筛选更大的化合物集合，以及产生许多候选分子， 筛选Prestwick和Broad的收藏品，以便在未来的实验中继续使用。

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.