Engineered Antibody EGFR Antagonist Cancer Therapeutics

描述(由申请人提供)：这是一个研究和对抗癌症中EGFR信号的多研究者项目的竞争性继续提案。研究人员利用了蛋白质工程(Wittrup，MIT)、质谱学磷酸蛋白质组学(White，MIT)、计算系统生物学(Lauffenburger，MIT)和结构生物学(Kuriyan，UC Berkeley)方面的专业知识。该项目的前十年共产生了52篇论文，共被引用2,088次，平均40篇，平均每篇论文被引用24次，h指数为26次。在这次更新中，我们建议扩展在上一次赠款期间开发的一种新的治疗方式，并加深我们对EGFR信号网络和受体结构/功能关系的理解。我们通过将两个基于Fn3的小的EGFR结合域添加到西妥昔单抗IgG上，创建了一种新的三表位抗体拓扑结构。这个单一的结构结合在EGFR上的三个不重叠的表位上，驱动快速聚集和下调，而没有检测到受体磷酸化或下游信号。三表位抗体控制耐药的移植瘤的生长 亲本西妥昔单抗，表明克服KRAS和BRAF突变引起的耐药性的作用机制有了质的改善。我们将扩展这种三表位结构的功能来抑制HER3，以便先发制人地克服一个关键的抗性机制。我们还将使用一种新型的EGFR靶向siRNA递送载体来鉴定沉默产生与EGFR拮抗协同作用的抗肿瘤效果的基因。我们将应用我们复杂的实验和计算网络分析工具来了解抗EGFR治疗药物的三种耐药形式：a)KRAS和BRAF等效应器激酶的突变；b)MET和HER3的上调；以及c)ErbB配体和胞外结构域的蛋白分解脱落的改变。在每种情况下，信号网络的互连性和动力学将在未治疗、西妥昔单抗治疗和三表位抗体治疗的细胞系中进行研究，以检查治疗干预措施如何与癌细胞中存在的失调通路相互作用。我们将扩展我们对EGFR受体生物学的结构研究，以加深我们对从细胞外EGFR配体结合到细胞质中的激酶激活的信息流的理解。膜蛋白核磁共振揭示了EGFR跨膜结构域的关键构象状态。结晶学、质谱学和荧光显微镜分析将有助于确定激活的EGFR二聚体中自动磷酸化的时间顺序和拓扑控制。这个异常全面整合的多学科项目势头极佳，团队正热情投入到这些激动人心的新方向上。

DESCRIPTION (provided by applicant): This is a competing continuation proposal for a multi-investigator project to study and contravene EGFR signaling in cancer. The investigators bring to bear expertise in protein engineering (Wittrup, MIT), mass spectrometric phosphoproteomics (White, MIT), computational systems biology (Lauffenburger, MIT), and structural biology (Kuriyan, UC Berkeley). The first ten years of this project has produced 52 publications that have been cited in total 2,088 times, with a mean of 40 and a median of 24 citations per paper, and an h index of 26. In this renewal we propose to extend a novel therapeutic modality developed in the previous grant period, and to deepen our understanding of EGFR signaling networks and receptor structure/function relationships. We have created a novel triepitopic antibody topology by appending two small Fn3-based EGFR binding domains to the cetuximab IgG. This single construct binds at three nonoverlapping epitopes on EGFR, driving rapid clustering and downregulation without detectable receptor phosphorylation or downstream signaling. The triepitopic antibody controls growth of xenografted tumors that are resistant to the parent cetuximab antibody, indicating a qualitative improvement in mechanism of action that overcomes resistance due to KRAS and BRAF mutations. We will extend the functionality of this triepitopic construct to inhibit HER3 in order to pre-emptively overcome a key resistance mechanism. We will also employ a novel EGFR-targeted siRNA delivery vector to identify genes whose silencing produce antitumor efficacy synergistic with EGFR antagonism. We will apply our sophisticated experimental and computational network analysis tools to understanding three forms of resistance to anti-EGFR therapeutics: a) mutations in effector kinases such as KRAS and BRAF; b) upregulation of MET and HER3; and c) altered proteolytic shedding of ErbB ligands and ectodomains. In each case, signaling network interconnectivity and dynamics will be studied in untreated, cetuximab treated, and triepitopic antibody treated cell lines to examine how therapeutic interventions interact with the dysregulated pathways present in cancer cells. We will extend our structural studies of EGFR receptor biology to deepen our understanding of the flow of information from EGFR ligand binding outside the cell to kinase activation in the cytoplasm. Membrane protein NMR is revealing key conformational states of the EGFR transmembrane domain. Crystallographic, mass spectrometric, and fluorescence microscopic assays will help identify the temporal order and topological control of autophosphorylation in the activated EGFR dimer. This unusually comprehensively integrated multidisciplinary project has excellent momentum, and the team is enthusiastically engaged in pressing forward in these exciting new directions.