Develop a rapid, efficient and low cost extracellular vesicles isolation method toward non-invasive non-small cell lung cancer molecular analysis

摘要/摘要 癌症是美国主要的死亡原因之一。据估计，大约有56.48万人死于 2017年癌症，每天有1500多人患癌症。高死亡率是缺乏营养的直接结果。 有效的早期临床诊断方法和治疗分层。如果癌症能够被有效地检测出来 早期治疗，如果治疗得当，平均五年相对生存率将大大提高。 胞外囊泡(EVS)是一种双层纳米大小的囊泡，存在于所有体液中，是一种 吸引人的癌症生物标志物，用于非侵袭性癌症诊断和治疗分层。电动汽车会 为需要重复组织活检的癌症患者提供显著的潜在益处。例如，SOLID 在大约49%的晚期或转移性非小细胞肺癌(NSCLC)患者中，肿瘤是无法触及的。 为了释放电动汽车在临床上的巨大应用潜力，快速高效地分离电动汽车是前提。 然而，目前的大多数EVS分离方法由于固有的原因不能满足临床的需要 缺点(例如，程序长、成品率低、吞吐量低)。此外，在临床应用方面 对于EV在癌症分子分析中的应用，目前还没有FDA批准的基于EV的诊断工具。卡普蒂斯 诊断公司开发了一种新的专有脂质纳米探针(LNP)系统，可以直接将EV从 样品(美国申请号62/430,161和国际申请号美国2017/064634)。LNP是 能够有效地将EV从细胞培养液或人体体液(例如血浆)中分离出来 工作流程和廉价试剂。在这项提案中，我们建议开发和商业化我们的脂肪 EV血浆分离纳米探针系统的发明和EV DNA分析平台的进一步开发 非小细胞肺癌患者的靶向治疗选择。对于目标1，Captis Diagnostics将优化和验证 LNP技术将其开发成可实现高EV隔离效率的EV隔离研究试剂盒， 工作流程简单快捷，能适应复杂的体液样本，如血浆。目标2将 建立用于非小细胞肺癌患者靶向治疗选择的概念验证性临床试验 应用数字滴状聚合酶链式反应(DdPCR)检测肠道病毒LNP分离株的突变。圆满完成这些目标 将产生专有的先进技术并定位Captis Diagnostics以追求临床验证 EV在癌症患者靶向治疗选择中的临床应用第二阶段的活动将集中在 非小细胞肺癌靶向治疗选择试验的临床表现，其中EV分离技术将 用癌症患者的样本进行测试，以验证其临床实用价值。

SUMMARY/Abstract Cancer is one of the leading causes of death in US. It is estimated that there were about 564,800 deaths from cancer in 2017, which was more than 1,500 people per day. The high mortality rate is a direct result of lack of effective early stage clinical diagnosis method and therapeutic stratification. If cancer can be effective detected early and treated with right therapy, the average five years relative survival rate would greatly increase. Extracellular vesicles (EVs), bilayered nano-sized vesicles that are found in all bodily fluids, are emerging as a appealing type of cancer biomarker for non-invasive cancer diagnosis and therapeutic stratification. EVs would provide significant potential benefit for cancer patients that need repetitive tissue biopsy. For example, solid tumors are not accessible in about 49% of advanced or metastatic non-small cell lung cancer (NSCLC) patients. To unleash EVs’ great potential in clinical applications, rapid and efficient isolation of EVs is a prerequisite. However, most of current EVs isolation approaches cannot meet the need in clinical settings due to inherent shortcomings (e.g. lengthy procedure, low yield, and low throughput). Additionally, in terms of clinical application of EV in cancer molecular analysis, there is no FDA approved EV-based diagnostic tools available. Captis Diagnostics has developed a new proprietary lipid nanoprobe (LNP) system that can isolate EVs directly from samples (US Application No. 62/430,161 and International Application No. PCT/US2017/064634). The LNP is capable of efficiently isolating EVs from cell culture media or human body fluids (e.g. plasma) using a rapid workflow and inexpensive reagents. In this proposal, we propose to develop and commercialize our lipid nanoprobe system invention for EV isolation from plasma and further develop EV DNA analysis platform for targeted therapy selection in NSCLC patients. For Aim 1, Captis Diagnostics will optimize and validate of the LNP technology to develop it to a research kit for EV isolation that can achieve high EV isolation efficiency, simple and short workflow, and adaption to complex bodily fluids samples, such as blood plasma. Aim 2 will develop a proof-of-concept clinical assay for targeted therapy selection in NSCLC patients by combining mutation detection by digital droplet PCR (ddPCR) with LNP EV isolation . Successful completion of these Aims will yield a proprietary advanced technology and position Captis Diagnostics to pursue clinical validation of the EV in clinical assay for targeted therapy selection for cancer patients. Phase II activities will be focused on the clinical performance of NSCLC targeted therapy selection assays, in which the EV isolation technology will be tested with samples from cancer patients to validate its clinical utility.