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 细胞外蛋白分解是癌症的一个标志。蛋白水解酶在功能上对 重塑细胞表面蛋白和基质，包括受体的激活和脱落 胞外结构域。在600多种人类蛋白酶中，超过一半存在于细胞表面或 分泌反映了它们对处理细胞外空间的意义。然而，我们非常理解 关于细胞表面蛋白分解(CSP)的靶点，负责的蛋白水解酶，以及由此产生的新的 裂解产物可以作为新的生物标记物或药物靶标。 缺少的是用于无偏见地识别CSP目标的稳健技术， 它们的特定切割位置，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的多肽连接酶，称为枯草杆菌酶， 并显示它有力地标记了可溶蛋白质的蛋白分解位点。我们的初步数据显示，这 可以使用强大的工具来确定CSP的切割靶点和精确的切割位置。我们建议 这项新的CSP技术可以用来确定癌症中的CSP信号。我们首先将重点放在 在KRAS上，最显性的癌基因，特别是在胰腺癌中。我们将确定KRAS- 低氧条件下培养的含突变和不含突变KRAS的同基因胰腺细胞系的蛋白分解 以及在二维和三维文化背景下的常氧条件。我们将确定 利用细胞表面捕获蛋白质组学上调表面蛋白水解酶及其活性 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性 将噬菌体技术与NGS相结合，并将这些与我们发现的新表位进行匹配。最后，我们将 进一步开发差别化抗体噬菌体展示筛选技术，产生特异性抗体 包括CDCP1在内的各种已识别靶标的切割形式。我们将发展重要的 新表位生物标记物技术和潜在的免疫肿瘤学试剂 这些重要的卵裂事件的特征。 影响：我们的研究将为公正的基于蛋白质工程的技术提供强大的 对CSP如何重塑细胞、负责的和可操作的癌基因特异性的蛋白的洞察 新表位产生抗体作为潜在的生物标记物和治疗学。我们相信这些 这种方法将普遍适用于描述其他实体瘤肿瘤中的蛋白分解。

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.