Immobilized protease activity tests for developing functional cancer biomarkers.

用于开发功能性癌症生物标志物的固定化蛋白酶活性测试。

• 批准号: 8847684
• 负责人: PAUL TEMPST
• 金额: $ 26.78万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-01 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8847684
• 关键词: Affect; Affinity; Aminopeptidase; Antibodies; Benign; Biological; Biological Assay; Biological Markers; Biopsy; Blood; Breast; Breast Cancer Detection; Breast biopsy; Cancer Patient; Caspase; Cathepsins; Cell Count; Cessation of life; Clinical; Clinical Chemistry; Data; Detection; Development; Diagnostic; Discrimination; Early Diagnosis; Enzyme-Linked Immunosorbent Assay; Enzymes; Fluorescence; Fractionation; Future; Gene Expression; Health; Hematopoietic Neoplasms; Histocytochemistry; Human; Immune; Individual; Liquid substance; MMP9 gene; Magnetic nanoparticles; Malignant - descriptor; Malignant Neoplasms; Malignant neoplasm of prostate; Mass Spectrum Analysis; Matrix Metalloproteinases; Measurement; Measures; Mediating; Metastatic Prostate Cancer; Methods; Modeling; Monitor; Neoplasm Circulating Cells; Noninfiltrating Intraductal Carcinoma; Outcome; Patients; Pattern; Peer Review; Peptide Hydrolases; Peptides; Performance; Plasma; Probability; Procedures; Prognostic Factor; Proteins; Proteome; Proteomics; Reading; Reagent; Recombinants; Research; Review Literature; Risk; Robot; Sampling; Scheme; Sensitivity and Specificity; Series; Serum; Shapes; Solid Neoplasm; Specificity; Techniques; Technology; Testing; Time; Tissues; Validation; Woman; advanced disease; base; cancer diagnosis; cancer risk; castration resistant prostate cancer; chemotherapy; clinically relevant; cohort; cost; follow-up; high risk; high throughput analysis; improved; inhibitor/antagonist; innovation; interest; malignant breast neoplasm; member; novel; portability; prognostic; prognostic tool; screening; success; technology development; tool

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.

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感兴趣组织中蛋白质的差异水平。这种方法并没有取得很大的成功，主要是因为血液蛋白质组的复杂性阻碍了检测低ng/mL水平的蛋白质或肽，而不需要耗时的预分离。这种现状可以通过改进现有标记或采用概念新颖的发现和验证方案来结束。为此，我们将开发一种创新的筛选和验证平台，使用新颖而稳健的方法来测量血液中的蛋白酶活性。这种方法背后的想法源于一种观察，即蛋白酶活动独特地塑造了血清肽丘，从而为不同类型实体瘤患者提供了类别区分。这一模型得到了大量同行评审文献的进一步支持，这些文献描述了实体肿瘤和血液中氨基肽酶和其他蛋白酶的基因表达、蛋白质水平和酶活性。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，在癌症患者的血液中似乎不平衡。与大多数传统的蛋白质测量相比，荧光分析具有优势，包括低成本，平行高通量分析，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，一种称为固定化蛋白酶活性测试（IPAT）的技术，显著提高了分析的特异性和敏感性。我们建议采购和测试关键试剂，以开发、优化和实施18种氨基肽酶和14种选定的其他蛋白酶（如MMPs、组织蛋白酶、半胱天冬酶等）的敏感IPAT测定。这样就可以测量癌症导致的血液中它们活动的定量变化。IPAT技术将在两种配置中实现：(1)抗体将固定在磁性纳米颗粒上，所有步骤在机器人中自动化（“头- IPAT”）。(2)将抗体固定在96孔微量滴度板（“well-IPAT”）中，配置类似于ELISA。结果集将在两个临床相关的生物标志物发现研究中进行测试。首先，为了评估血浆蛋白酶检测作为提高乳腺癌放射筛查特异性手段的效用，我们将评估其在预测乳腺活检结果方面的表现。其次，我们将检验血清IPAT测定在接受化疗的进展性转移性去势抵抗性前列腺癌患者中作为预后因素的价值。临床目标是将这些数据与现有的（尽管不完善）标志物结合起来，开发出一种准确给出患者生存概率的预后工具。这一应用具有创新性，因为这是第一次将一类酶作为潜在活性生物标志物，而这些酶在过去的蛋白质组学筛选中未被检测到。如果我们的研究取得成功，并因此产生有效的功能性癌症生物标志物面板，它将产生重大的临床影响。