Intramolecular Immunoassay for Probing Paracrine Signaling

描述（由申请人提供）：肿瘤发生的关键要素由细胞外基质背景下基质细胞和上皮细胞之间的旁分泌信号传导驱动。不幸的是，用于药物发现的细胞测定通常依赖于作为单层生长的单细胞类型，并且这方面的肿瘤生物学非常差。这可能是导致抗癌药物在临床试验中成功率低的一个因素。虽然在组织样环境中包含多种细胞类型的细胞模型是可用的，但它们难以与制药公司用于细胞功能的高含量分析的当前自动化显微成像平台整合。一个特别具有挑战性的问题是检测细胞外基质中的可溶性旁分泌信号传导因子。尽管抗体可用于许多这些因素，但现有的免疫测定方法不适合在密集基质中使用。一个根本的限制是需要多个组分：一抗和二抗报告试剂。为了克服这一限制，我们提出开发分子内免疫测定;即，一种内置荧光报告基因的抗体我们将通过将柔性多肽接头上的荧光示踪剂拴系到用硒代半胱氨酸残基工程化的IgG和Fab片段以进行位点特异性修饰来实现这一点。将使用血管内皮生长因子（VEGF）的良好表征的单克隆抗体作为初始模型。将组装一系列原型分子内免疫测定试剂，并测试基于荧光的VEGF检测，以达到最佳结构。在第二阶段，分子内免疫测定将与BellBrook的新型微导管阵列平台iuvo相结合，以实现在单个细胞分泌的可溶性因子水平上对旁分泌信号的动态成像。 公共卫生相关性： 肿瘤发生的重要方面是由不同细胞类型之间的信号传导控制的，这些细胞类型位于为组织提供结构支持并参与信号传导的致密基质中。不幸的是，很难将这些信号事件纳入可用于测试潜在抗癌药物的体外测定中。为了克服这一局限性，我们建议开发一种新的免疫测定方法，该方法将允许使用现有的自动检测仪器探测组织样基质中的细胞-细胞信号。

DESCRIPTION (provided by applicant): Key elements of tumorigenesis are driven by paracrine signaling between stromal and epithelial cells within the context of the extracellular matrix. Unfortunately, the cellular assays used for drug discovery typically rely on a single cell type grown as a monolayer, and this aspect of tumor biology very poorly. This may be one factor contributing to the low success rate of anti-cancer drugs in clinical trials. Though cellular models incorporating multiple cell types in a tissue-like environment are available, they are difficult to integrate with current automated microscopic imaging platforms used by pharma for high content analysis of cell function. An especially challenging problem is detection of soluble paracrine signaling factors in extracellular matrix. Though antibodies are available for many of these factors, existing immunoassay methods are not suited for use in a dense matrix. A fundamental limitation is the requirement for multiple components: the primary antibody and secondary reporter reagents. To overcome this limitation we are proposing to develop an intramolecular immunoassay; i.e., an antibody with a built-in fluorescent reporter. We will achieve this by tethering a fluorescent tracer on a flexible polypeptide linker to IgG and Fab fragments engineered with seleno cysteine residues for site specific modification. A well characterized monoclonal antibody for vascular endothelial growth factor (VEGF) will be used as an initial model. A series of prototype intramolecular immunoassay reagents will be assembled and tested for fluorescence based detection of VEGF to arrive at the optimal structure. In Phase II, the intramolecular immunoassay will be combined with BellBrook's novel microconduit array platform, iuvo, to enable dynamic imaging of paracrine signaling at the level of soluble factors secreted from individual cells. PUBLIC HEALTH RELEVANCE: Important aspects of tumorigenesis are controlled by signaling between different cell types that reside in a dense matrix that provides structural support to tissues and also participates in signaling. Unfortunately, it is very difficult to incorporate these signaling events into in vitro assays that can be used to test potential anti-cancer drugs. To overcome this limitation, we propose to develop a novel immunoassay method that would allow probing of cell-cell signaling in a tissue-like matrix using existing automated detection instruments.