Approaches to discover and quantify apoptotic biomarkers for cancer treatment

描述(由申请人提供)：蛋白分解途径在几乎所有人类疾病中都是突出的，是从分化到死亡的基本细胞功能的共同特征。这项建议的长期目标是开发一种新的、快速的、定量的平台来监测血液肿瘤化疗患者中与细胞凋亡相关的蛋白分解活性。我们假设，caspase蛋白分解的产物从死亡的凋亡癌细胞释放到循环中，这些标记的一个子集将是细胞类型特异性的，因此是恶性肿瘤特异性的。一种监测化疗的血清生物标记物方法代表了癌症临床治疗的范式转变，这主要依赖于放射成像和实验室测试。因为这些研究通常在治疗开始后几周到几个月进行，所以经常进行不必要的或无效的化疗。由于目前用于识别、检测和定量蛋白水解性片段的方法繁琐、吞吐量低且成本高昂，我们建议开发一个包括假说驱动的发现方法、新的新表位靶向抗体和新的多重定量方法的平台。为了识别这些凋亡生物标记物，我们将开发有针对性的质谱学方法，在恶性血液病细胞培养模型和接受化疗的这些癌症患者的血浆中鉴定caspase活性诱导凋亡时产生的蛋白分解产物(目标1)。这种方法允许一种史无前例的大规模有针对性的方法来检测和分层血清中的凋亡生物标记物，可以用来监测接受化疗的患者的治疗情况。然而，目前缺乏可靠和高通量的方法来测量血浆中蛋白水解性衍生生物标记物的水平，这阻碍了这种实时策略，因此，我们将开发针对caspase在细胞凋亡过程中切割的蛋白质的新表位的抗体(目标2)。这里开发的基于噬菌体的方法将能够以多参数的方式对凋亡生物标记物进行量化，并将费力且往往不确定的产生高亲和力和特异性的单抗的过程转变为定义明确、廉价和可再生的过程。最后，我们将开发独特的生物标记物定量方法，包括一种新的高度多重噬菌体和下一代测序分析(PHANGS)(目标3)。在弥漫性大B细胞淋巴瘤和多发性骨髓瘤患者的治疗反应与生物标记物水平相关的先导性研究中应用这些方法，将为进一步的生物标记物验证研究提供必要的基础。这些途径和方法具有广泛的生物医学应用，包括单细胞和动物的生物成像和蛋白分解检测，以及新表位抗体的新的治疗用途。此外，虽然这些工具和技术专注于血液恶性肿瘤，但它们将显著阐明蛋白质分解在一系列人类疾病中的关键作用，如癌症、传染病和神经退行性变。 公共卫生相关性：拟议的研究将导致开发一种血清生物标记物方法，以实现对化疗反应和疗效的快速和实时监测，这将代表着癌症临床管理的重大进步。此外，这里开发的新技术将极大地促进其他人类疾病中蛋白质分解的研究。

DESCRIPTION (provided by applicant): Proteolytic pathways are prominent in virtually all human diseases and are common hallmarks of basic cellular functions ranging from differentiation to death. The long-term goal of this proposal is to develop a novel, rapid, quantitative platform to monitor the proteolytic activity associated with apoptosis in patients undergoing chemotherapy for hematologic malignancies. We hypothesize that products of caspase proteolysis are released from dying apoptotic cancer cells into the circulation and that a subset of these markers will be cell type-specific, and therefore malignancy-specific. A serum biomarker approach to monitoring chemotherapy represents a paradigm shift in clinical management of cancer, which mostly relies upon radiographic imaging and laboratory tests. Because these studies are typically performed weeks to months after the start of therapy, unnecessary or ineffective chemotherapy is often administered. Since current methods to identify, detect, and quantify proteolytic fragments are cumbersome, low-throughput, and costly, we propose to develop a platform that involves a hypothesis-driven discovery method, novel neo-epitope targeted antibodies, and a new multiplexed quantification method. To identify these apoptotic biomarkers, we will develop targeted mass spectrometric methods for identifying proteolysis products created by caspase activity upon induction of apoptosis in cell culture models of hematologic malignancies and plasma of patients undergoing chemotherapy for these cancers (Aim 1). This approach permits an unprecedented large-scale targeted way to detect and stratify apoptotic biomarkers in serum that can be used to monitor treatment of patients undergoing chemotherapy. However, the current lack of reliable and high-throughput methods to measure proteolytically- derived biomarker levels in plasma prevents such a real-time strategy and thus, we will develop antibodies specifically directed against the neo-epitopes of proteins cleaved by caspases during apoptosis (Aim 2). The phage-based methods developed here will enable quantification of apoptotic biomarkers in a multi-parameter fashion and transform the laborious and often uncertain process of generating monoclonal antibodies with high affinities and specificities into a well-defined, cheap, and renewable one. Finally, we will develop unique methods for biomarker quantification, including a novel highly multiplexed phage and next generation sequencing assay (PHANGS) (Aim 3). Application of these methods in a pilot study to correlate biomarker levels to treatment response in patients with diffuse large B-cell lymphoma and multiple myeloma will provide essential groundwork to further biomarker validation studies. These approaches and methods have vast biomedical applications, including biological imaging and detection of proteolysis in single cells and animals as well as novel therapeutic uses of neo-epitope antibodies. Furthermore, while focused on hematologic malignancies, these tools and technologies will significantly elucidate the crucial role of proteolysis in a spectrum of human diseases, such as cancer, infectious disease, and neurodegeneration. PUBLIC HEALTH RELEVANCE: The proposed study will result in the development of a serum biomarker approach to permit rapid and real-time monitoring of chemotherapeutic response and efficacy that will represent a significant advance in clinical management of cancer. Furthermore, the novel technologies developed here will significantly advance the study of proteolysis in other human diseases.