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

项目摘要 细胞外蛋白水解是癌症的标志。蛋白质水解具有主要的功能性后果， 重塑细胞表面蛋白和基质，包括受体活化和受体脱落 胞外结构域。在超过600种人类蛋白酶中，超过一半位于细胞表面或 分泌反映其对处理细胞外空间的重要性。但我们非常理解 关于细胞表面蛋白水解（CSP）的靶点，负责的蛋白酶，以及由此产生的新- 裂解产物可以作为新的生物标志物或药物靶点。 缺少的是用于无偏见地识别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 新表位特异性抗体。我的团队已经设计了一种新的肽连接酶，叫做枯草杆菌连接酶， 并显示它能有力地标记可溶性蛋白质的蛋白水解位点。我们的初步数据显示， 这是一个强有力的工具，可用于确定CSP的靶点和精确切割位点。我们提出 这种新的CSP技术可用于确定癌症中的CSP特征。我们将首先关注 KRAS是最主要的致癌基因，尤其是在胰腺癌中。我们将决定KRAS- 在低氧环境中生长的具有和不具有突变KRAS的同基因胰腺细胞系中诱导蛋白水解 和常氧条件下的二维和三维培养。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。我们将使用新的底物来表征它们的特异性 噬菌体技术与NGS结合，并将这些与我们发现的新表位相匹配。最后，我们将 进一步利用差异抗体噬菌体展示筛选技术， 包括CDCP 1在内的各种已鉴定靶点的切割形式。我们将发展重要的 新的新表位生物标志物和潜在的免疫肿瘤学试剂的技术， 这些重要的裂解事件的表征。 影响：我们的研究将提供强大的基于蛋白质工程的技术， 深入了解CSP如何重塑细胞，负责的蛋白酶和可操作的癌基因特异性 新表位以产生作为潜在生物标志物和治疗剂的抗体。我们相信这些 这些方法通常可用于表征其它实体瘤癌症中的蛋白水解。

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.