New protein engineering-based tools and technologies for characterizing cell surface proteolysis in cancer cells for novel neo-epitope biomarkers and drug targets

基于新蛋白质工程的工具和技术，用于表征癌细胞中的细胞表面蛋白水解，以获得新型新表位生物标志物和药物靶点

基本信息

批准号：
10582604
负责人：
JAMES A WELLS
金额：
$ 35.61万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-04-01 至 2025-03-31
项目状态：
未结题

项目摘要

Project Summary Extracellular proteolysis is a hallmark of cancer. Proteolysis has major functional consequences for remodeling cell surface proteins and matrix including receptor activation and shedding of receptor extracellular domains. Of the more than 600 human proteases, over half are on the cell surface or secreted reflecting their significance for processing the extracellular space. Yet we understand very little about the targets for cell surface proteolysis (CSP), the proteases responsible, and resulting neo- cleaved products that could serve as new biomarkers or drug targets. What has been missing are robust technologies for unbiased identification of the CSP targets, their specific sites of cleavage, the proteases responsible for promoting disease, and the generation of neo-epitope specific antibodies. My group has engineered a novel peptide ligase, called subtiligase, and shown it robustly labels sites of proteolysis of soluble proteins. Our preliminary data show that this powerful tool can be used to determine the targets and precise sites of cleavage for CSP. We propose this novel CSP technology can be used to determine CSP signatures in cancer. We will initially focus on KRAS, the most dominant oncogene, especially in pancreatic cancer. We will determine KRAS- induced proteolysis in isogenic pancreatic cell lines, with and without mutant KRAS, grown in hypoxic and normoxic conditions in the context of two- and three-dimensional culture. We will identify the surface proteases up-regulated using cell surface capture (CSC) proteomics and confirm their activities using CRISPRi or small molecule inhibitors. We will characterize their specificities using new substrate phage technology coupled with NGS and match these to neo-epitopes we discover. Lastly, we will further exploit differential antibody phage display selection technology, and generate antibodies specific to the cleaved forms of various identified targets, including CDCP1. We will develop important technologies for new neo-epitope biomarkers and potential immune-oncology reagents for the characterization of these important cleavage events. Impact: Our studies will provide robust protein engineering-based technologies for unbiased insights into how CSP remodels cells, the proteases responsible, and actionable oncogene specific neo-epitopes to generate antibodies as potential biomarkers and therapeutics. We believe these approaches will be generally applicable to characterizing proteolysis in other solid tumor cancers.

项目概要细胞外蛋白水解是癌症的标志。蛋白水解具有主要的功能后果重塑细胞表面蛋白和基质，包括受体激活和受体脱落细胞外结构域。在 600 多种人类蛋白酶中，一半以上位于细胞表面或分泌反映了它们对处理细胞外空间的重要性。然而我们却非常理解关于细胞表面蛋白水解（CSP）的靶标、负责的蛋白酶以及由此产生的新蛋白知之甚少。可以作为新的生物标志物或药物靶标的裂解产物。所缺少的是用于公正识别 CSP 目标的强大技术，它们的特定切割位点、负责促进疾病的蛋白酶以及产生新表位特异性抗体。我的团队设计了一种新型肽连接酶，称为枯草连接酶，并表明它可以强有力地标记可溶性蛋白质的蛋白水解位点。我们的初步数据表明，这强大的工具可用于确定 CSP 的目标和精确的切割位点。我们建议这种新颖的 CSP 技术可用于确定癌症中的 CSP 特征。我们首先将重点 KRAS 是最主要的癌基因，尤其是在胰腺癌中。我们将确定 KRAS- 在含或不含突变型 KRAS 的同基因胰腺细胞系中诱导蛋白水解，在低氧条件下生长二维和三维文化背景下的含氧量正常条件。我们将确定使用细胞表面捕获 (CSC) 蛋白质组学上调表面蛋白酶并确认其活性使用 CRISPRi 或小分子抑制剂。我们将使用新基材来表征它们的特性噬菌体技术与 NGS 相结合，并将其与我们发现的新表位相匹配。最后，我们将进一步利用差异化抗体噬菌体展示选择技术，产生特异性抗体各种已识别靶标的切割形式，包括CDCP1。我们将开发重要的新表位生物标志物技术和潜在的免疫肿瘤学试剂这些重要裂解事件的表征。影响：我们的研究将为公正的研究提供基于蛋白质工程的强大技术深入了解 CSP 如何重塑细胞、负责的蛋白酶以及可操作的癌基因特异性新表位产生抗体作为潜在的生物标志物和治疗剂。我们相信这些这些方法将普遍适用于表征其他实体瘤癌症中的蛋白水解。