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

概要/摘要 癌症是美国主要的死亡原因之一。据估计，约有564，800人死于 2017年，每天有超过1,500人患上癌症。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。如果癌症能被有效地检测出来 早期正确治疗可大大提高平均5年相对生存率。 细胞外囊泡（EV），在所有体液中发现的双层纳米大小的囊泡，正在成为一种新的生物学机制。 对于非侵入性癌症诊断和治疗分层来说，这是一种有吸引力的癌症生物标志物。EV将 为需要重复组织活检的癌症患者提供显著的潜在益处。例如固体 在约49%的晚期或转移性非小细胞肺癌（NSCLC）患者中，肿瘤是不可接近的。 为了释放EV在临床应用中的巨大潜力，快速有效地分离EV是先决条件。 然而，由于固有的缺陷，目前大多数EV隔离方法不能满足临床环境中的需要。 缺点（例如，冗长的程序、低产量和低通量）。此外，在临床应用方面， 尽管EV在癌症分子分析中的应用非常广泛，但目前还没有FDA批准的基于EV的诊断工具可用。卡皮斯 诊断开发了一种新的专有脂质纳米探针（LNP）系统，可以直接从 样品（美国申请号62/430，161和国际申请号PCT/US 2017/064634）。LNP是 能够使用快速分离技术从细胞培养基或人体液（例如血浆）中有效分离EV， 工作流程和廉价的试剂。在这项提案中，我们建议开发和商业化我们的脂质， 发明了用于从血浆中分离EV的纳米探针系统，并进一步开发了EV DNA分析平台， NSCLC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，将其发展为可实现高EV隔离效率的EV隔离研究套件， 简单和短的工作流程，并适应复杂的体液样品，如血浆。目标2将 开发一种用于NSCLC患者靶向治疗选择的概念验证临床试验， 通过数字液滴PCR（ddPCR）和LNP EV分离进行突变检测。成功实现这些目标 将产生一种专有的先进技术，并使Captis Diagnostics能够对 EV在癌症患者靶向治疗选择的临床检测中的应用。第二阶段的活动将侧重于 NSCLC靶向治疗选择试验的临床性能，其中EV分离技术将 用癌症患者的样本进行测试，以验证其临床效用。

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.