Immobilized protease activity tests for developing functional cancer biomarkers.

描述（由申请人提供）：发现蛋白质生物标志物的经典途径是通过测量血液或组织中蛋白质的差异水平。这种方法尚未取得巨大成功，主要是因为血液蛋白质组的复杂性排除了在没有耗时的预分级分离的情况下检测低ng/mL水平的蛋白质或肽。这种现状可能会结束，要么改善现有的标记或采用发现和验证方案，在概念上是新颖的。为此，我们将开发一个创新的筛选和验证平台，使用新颖而强大的方法来测量血液中的蛋白酶活性。这种方法背后的想法起源于一项观察，即蛋白酶活性独特地塑造血清肽组，以提供不同类型实体瘤患者之间的类别区分。该模型得到了大量同行评议文献的进一步支持，这些文献描述了实体瘤和血液中氨肽酶和其他蛋白酶的基因表达、蛋白水平和酶活性。它为生物标志物的发现和验证方法提供了机会，这些方法在今天的标准下是独一无二的。我们之前已经开发了一系列基于荧光的活性测定来分析单个蛋白酶，而不是过去测量的集体活性，这些活性在癌症患者的血液中似乎是不平衡的。荧光测定具有优于大多数常规蛋白质测量的优点，包括低成本、并行高通量分析、易于使用、便携性以及对整个血液蛋白质组完全透明。通过将抗体（Ab）介导的亲和捕获步骤放在前面，称为固定化蛋白酶活性测试（IPAT）的技术，显著提高了分析特异性和灵敏度。我们建议采购和测试关键试剂，以开发、优化和实施针对18种氨肽酶和14种选定的其他蛋白酶（例如，MMP、组织蛋白酶、半胱天冬酶等）。这将允许测量由于癌症而导致的血液中的活性的定量变化。IPAT技术将以两种配置实施：（1）Ab将被固定到磁性纳米颗粒上，并且所有步骤在机器人中自动化（“珠-IPAT”）。(2)以类似于ELISA的配置固定在96孔微量滴定板（“孔-IPAT”）中的Ab。将在两项临床相关生物标志物发现研究中对所得数据集进行检测。首先，为了评估血浆蛋白酶测定作为提高乳腺癌放射学筛查特异性的一种手段的实用性，我们将评估其在预测乳腺活检结果方面的性能。其次，我们将研究血清IPAT测定作为接受化疗的进展性转移性去势抵抗性前列腺癌患者生存的预后因素的价值。临床目标是将联合收割机的数据与现有的，但不完善的标记物相结合，并开发一种准确给出患者生存概率的预后工具。这一应用是创新的，因为它是第一次一类酶，在过去的蛋白质组学筛选中未被检测到，将被靶向作为潜在的活性生物标志物。如果我们的开发成功，并因此出现有效的功能性癌症生物标志物小组，它将产生重大的临床影响。

DESCRIPTION (provided by applicant): The classic path to protein biomarker discovery is by measuring differential levels of proteins in blood or tissues of interest. This approach has not been a great success, mainly because the complexity of the blood proteome precludes detection of proteins or peptides at low ng/mL levels without time-consuming pre- fractionation. This status quo may end by either improving existing markers or by employing discovery and validation schemes that are conceptually novel. To this end, we will develop an innovative screening and verification platform using novel yet robust approaches to measure protease activities in blood. The idea behind this approach originated in an observation that protease activities uniquely shape the serum peptidome to provide class discrimination between patients with different types of solid tumors. This model is further supported by a wealth of peer-reviewed literature describing gene expression, protein levels and enzymatic activities of aminopeptidases, and other proteases, in solid tumors and blood. It presents opportunities for biomarker discovery and validation approaches that are unique by today's standards. We have previously developed a series of fluorescence-based activity assays to analyze individual proteases, as opposed to the collective activities measured in the past, that appear unbalanced in blood of cancer patients. Fluorescent assays have advantages over most conventional protein measurements, including low cost, parallel high- throughput analysis, ease of use, portability, and being fully transparent to the entire blood proteome. By putting an antibody (Ab)-mediated affinity-capture step on the front, a technique termed Immobilized Protease Activity Test (IPAT), analytical specificity and sensitivity are significantly improved. We propose to procure and test critical reagents to develop, optimize and implement sensitive IPAT assays for 18 aminopeptidases and 14 selected other proteases (e.g., MMPs, cathepsins, caspases, etc). This will allow measuring quantitative changes of their activities in blood as a result of cancer. IPAT technology will be implemented in two configurations: (1) Abs will be immobilized to magnetic nanoparticles and all steps automated in a robot ('bead- IPAT'). (2) Abs immobilized in 96-well micro-titer plates ('well-IPAT') in a configuration similar to an ELISA. The resulting set will be tested in two clinically relevant biomarker discovery studies. First, to evaluate the utiliy of plasma protease assays as a means of improving specificity of radiographic screening for breast cancer, we will assess its performance in predicting the outcome of breast biopsies. Second, we will examine the value of serum IPAT assays as a prognostic factor for survival in patients with progressive metastatic castration- resistant prostate cancer receiving chemotherapy. The clinical objective is to combine the data with existing, though imperfect markers and develop a prognostic tool that accurately gives the probability of patient survival. This application is innovative as it is the first time a class of enzymes, undetected in past proteomic screens, will be targeted as potential activity biomarkers. If our developments are successful and, as a result, effective functional cancer biomarker panels emerge, it would have a major clinical impact.