Developing a screening campaign for immune enhancers

旨在增强免疫力的疗法正在成为癌症疗法的重要组成部分，并且可以 用于增强抗病毒免疫力并增强疫苗的功效。许多策略都集中在 生物制剂，例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，这种药物的其他药物 类型可能有用。小分子免疫增强剂如此之少的原因之一是 历史上一直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 偶然发现类似物可以增强免疫反应。 我们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。该测定是我们成功应用的 HTS 的强大修订版 到 NIH 的分子图书馆小分子存储库。在新的增强检测中，我们刺激 TALL- 104 个人类白血病细胞毒性 T 淋巴细胞 (CTL)，带有涂有抗 CD3 抗体的珠子，生成 特别是在珠子结合群体中，次最大胞吐作用。我们通过测量结合来监测胞吐作用 使用流式细胞术将针对 LAMP-1 (CD107a) 的荧光标记抗体注入细胞。这使我们能够 进行免洗测定，可以检测增强胞吐作用的化合物并将其与 本身引起胞吐作用的化合物。 TALL-104 CTL 在我们的战略中既作为 免疫相关细胞类型以及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。使用 TALL-104 细胞进行筛选，然后评估 命中对其他重要免疫功能的影响可能会导致治疗线索的识别或 可用于识别可用于产生免疫的新细胞靶标的探针 增强。 我们的三个目标是 1) 开发和优化一种高度复杂的检测方法，用于评估 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 化合物库优化测定； 2）开发一套辅助测试来检查 命中对 CTL 杀伤靶细胞的影响以及辅助 T 细胞、B 细胞和 树突状细胞和 3) 通过筛选 Broad Institute 的 DOS informer 来验证 HTS/后续策略 约 10K 化合物的集合。完成我们的目标将提供适合的检测和后续工作流程 筛选更大的化合物集合，以及生成许多候选分子 筛选可在未来实验中进行的普雷斯蒂克和布罗德收藏。

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.