Intramolecular Immunoassay for Probing Paracrine Signaling

描述(申请人提供)：肿瘤发生的关键因素是由细胞外基质中间质和上皮细胞之间的旁分泌信号驱动的。不幸的是，用于药物发现的细胞分析通常依赖于作为单层生长的单一细胞类型，而肿瘤生物学的这一方面非常糟糕。这可能是抗癌药物临床试验成功率低的原因之一。尽管在类似组织的环境中融合了多种细胞类型的细胞模型是可用的，但它们很难与目前制药公司用于高含量细胞功能分析的自动化显微成像平台相结合。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因子。虽然这些因素中的许多都有抗体，但现有的免疫分析方法不适合在密集的基质中使用。一个基本的限制是对多种成分的要求：一次抗体和二次报告试剂。为了克服这一限制，我们建议开发一种分子内免疫分析；即带有内置荧光报告的抗体。我们将通过将柔性多肽连接物上的荧光示踪剂拴在带有硒半胱氨酸残基的抗体和Fab片段上进行位点特异性修饰来实现这一点。一种特性良好的抗血管内皮生长因子(VEGF)的单抗将被用作初始模型。将组装一系列原型分子内免疫分析试剂并进行测试，以基于荧光检测血管内皮生长因子，以获得最佳结构。在第二阶段，分子内免疫分析将与BellBrook的新型微管阵列平台iuvo相结合，以实现在单个细胞分泌的可溶性因子水平上的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类型的细胞之间的信号控制的，这些细胞类型驻留在一个密集的基质中，为组织提供结构支持，并参与信号传递。不幸的是，将这些信号事件纳入可用于测试潜在抗癌药物的体外测试中是非常困难的。为了克服这一限制，我们建议开发一种新的免疫分析方法，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.