Intramolecular Immunoassay for Probing Paracrine Signaling

描述（由申请人提供）：肿瘤发生的关键因素是由细胞外基质中基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单层生长的单个细胞类型，这方面的肿瘤生物学非常差。这可能是导致抗癌药物临床试验成功率低的一个因素。虽然在类组织环境中包含多种细胞类型的细胞模型是可用的，但它们很难与制药公司用于高含量细胞功能分析的当前自动显微成像平台集成。一个特别具有挑战性的问题是检测可溶性旁分泌信号因子在细胞外基质。虽然抗体可用于许多这些因素，现有的免疫测定方法不适合在密集的基质中使用。一个基本的限制是需要多种成分：一抗和二级报告试剂。为了克服这一限制，我们建议开发一种分子内免疫测定法；也就是说，一种内置荧光报告基因的抗体。我们将通过将荧光示踪剂系在柔性多肽连接体上，连接到含有硒半胱氨酸残基的IgG和Fab片段，以进行位点特异性修饰。血管内皮生长因子（VEGF）单克隆抗体将被用作初始模型。我们将组装并测试一系列分子内免疫测定试剂的原型，用于基于荧光的VEGF检测，以获得最佳结构。在II期，分子内免疫分析将与BellBrook的新型微导管阵列平台iuvo相结合，以实现从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。

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.