Approaches to discover and quantify apoptotic biomarkers for cancer treatment

描述（由申请人提供）：蛋白水解途径在几乎所有人类疾病中都很突出，并且是从分化到死亡的基本细胞功能的共同标志。该提案的长期目标是开发一种新的，快速的，定量的平台来监测与接受化疗的血液恶性肿瘤患者的细胞凋亡相关的蛋白水解活性。我们假设，caspase蛋白水解的产物从死亡的凋亡癌细胞释放到循环中，并且这些标记物的子集将是细胞类型特异性的，因此是恶性肿瘤特异性的。监测化疗的血清生物标志物方法代表了癌症临床管理的范式转变，其主要依赖于放射成像和实验室检查。由于这些研究通常在治疗开始后数周至数月进行，因此通常会进行不必要或无效的化疗。由于目前的方法来识别，检测和定量蛋白水解片段是繁琐的，低通量，和昂贵的，我们建议开发一个平台，涉及一个假设驱动的发现方法，新的新表位靶向抗体，和一个新的多重定量方法。为了鉴定这些凋亡生物标志物，我们将开发靶向质谱方法，用于鉴定在恶性血液病细胞培养模型和接受这些癌症化疗的患者血浆中诱导凋亡时由胱天蛋白酶活性产生的蛋白水解产物（目的1）。这种方法允许一种前所未有的大规模靶向方法来检测和分层血清中的凋亡生物标志物，可用于监测接受化疗的患者的治疗。然而，目前缺乏测量血浆中蛋白水解衍生的生物标志物水平的可靠和高通量方法阻止了这种实时策略，因此，我们将开发特异性针对细胞凋亡期间被半胱天冬酶切割的蛋白质的新表位的抗体（目的2）。本文开发的基于噬菌体的方法将能够以多参数方式定量凋亡生物标志物，并将产生具有高亲和力和特异性的单克隆抗体的费力且通常不确定的过程转变为定义明确、廉价且可再生的过程。最后，我们将开发用于生物标志物定量的独特方法，包括新型高度多重噬菌体和下一代测序测定（PHANGS）（目标3）。在一项初步研究中应用这些方法将弥漫性大B细胞淋巴瘤和多发性骨髓瘤患者的生物标志物水平与治疗反应相关联，这将为进一步的生物标志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，包括单细胞和动物中蛋白水解的生物成像和检测以及新表位抗体的新治疗用途。此外，虽然专注于血液恶性肿瘤，这些工具和技术将显着阐明蛋白水解在一系列人类疾病中的关键作用，如癌症，传染病和神经变性。 公共卫生相关性：拟议的研究将导致血清生物标志物方法的发展，以允许快速和实时监测化疗反应和疗效，这将代表癌症临床管理的重大进展。此外，这里开发的新技术将大大推进其他人类疾病中蛋白质水解的研究。

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.