Engineering the Met receptor: a potent antagonist of tumor growth and metastasis

描述(由申请人提供)：Met受体酪氨酸激酶对调节细胞的增殖、存活和迁移至关重要。人类肿瘤侵袭和转移的趋势与Met的调节失调有关；因此，Met是一个极具吸引力的治疗干预靶点。有趣的是，可溶性Met胞外区(Met-ECD)是Met激活的有力拮抗剂。遗憾的是，目前重组表达Met-ECD的方法产量较低，这阻碍了其生物学特性的充分表征和作为潜在治疗手段的部署。我们的实验室拥有强大和成熟的技术来进化、优化和表达这种具有挑战性的蛋白质，Met-ECD是应用这项技术的理想候选者。这项工作将加速Met配体/受体相互作用的生物物理表征，以及Met受体在癌症治疗和诊断中的应用。目的1：设计全长Met胞外区，在酵母中高效可溶性表达。酵母表面展示的定向进化为我们提供了一个强大的组合平台来鉴定Met-ECD突变体，这些突变体1)具有天然受体折叠，2)在酵母中表现出高水平的可溶性表达(mg/L)。目的：检测Met-ECD突变体介导的配体结合亲和力和生物活性。我们将测试目标1中分离的Met-ECD突变体，以验证表达改善的工程受体保留了天然配体结合特性和生物学功能。我们将把Met-ECD突变体与野生型Met跨膜和胞内结构域融合，并将这些构建体导入表达低水平内源性Met的哺乳动物细胞系。我们将测试这些细胞系结合HGF配体和诱导细胞信号传递的能力，并将它们与野生型Met转基因细胞进行比较。这些努力将证明Met-ECD突变体的生物学功能，为未来的生物物理研究验证它们。目的3：检测可溶性Met-ECD突变体与肿瘤细胞结合并抑制信号转导的能力。我们将通过测量可溶性Met-ECD突变体是否具有受体拮抗剂的能力来确定它们是否具有受体拮抗剂的功能：1)与表达Met的肿瘤细胞结合；2)抑制Met激酶结构域及其下游MAPK和PI3K信号通路的配体依赖和配体非依赖的激活。这一目标将验证Met-ECD突变体在各种细胞类型中作为受体拮抗剂的有效性，并推动未来对其治疗潜力的研究。项目简介功能性重组Met-ECD的可用性将深刻影响未来对肿瘤发生、细菌发病和胚胎发育机制的研究，并将为如何操纵这些过程提供洞察力。Met-ECD本身也可以被开发用于分子成像或癌症治疗，这一事实是该项目影响巨大的另一个例子。

DESCRIPTION (provided by applicant): The Met receptor tyrosine kinase is critical for mediating cell proliferation, survival and migration. The tendency for human tumors to invade and metastasize has been tied to the dysregulation of Met; therefore, Met is an extremely attractive target for therapeutic intervention. Interestingly, soluble Met extracellular domain (Met-ECD) acts as a potent antagonist of Met activation. Unfortunately, current methods for recombinant expression of Met-ECD have low yield, preventing both the full characterization of its biology and its deployment as a potential therapeutic. Our lab has robust and proven technology for the evolution, optimization, and expression of such challenging proteins, and Met-ECD is an ideal candidate for applying this technology. This work will accelerate biophysical characterization of Met ligand/receptor interactions, and applications of the Met receptor in cancer therapy and diagnostics. Aim 1: Engineer the full-length Met extracellular domain for high soluble expression levels in yeast. Directed evolution using yeast surface display provides us with a robust combinatorial platform to identify Met- ECD mutants that 1) possess the native receptor fold and 2) exhibit high soluble expression levels (mg/L) in yeast. Aim 2: Measure the ligand binding affinity and biological activity mediated by Met-ECD mutants. We will test the Met-ECD mutants isolated in Aim 1 to verify that engineered receptors with improved expression retain native ligand binding properties and biological function. We will fuse Met-ECD mutants to wild-type Met transmembrane and intracellular domains, and transfect these constructs into a mammalian cell line that expresses low levels of endogenous Met. We will test these cells lines for their ability to bind HGF ligand and induce cell signaling, comparing them to cells transfected with wild-type Met. These efforts will demonstrate the biological functions of Met-ECD mutants, validating them for future biophysical studies. Aim 3: Determine the ability of soluble Met-ECD mutants to bind to tumor cells and inhibit signaling. We will determine if soluble Met-ECD mutants function as receptor antagonists by measuring their ability to 1) bind to Met-expressing tumor cells and 2) inhibit ligand-dependent and ligand-independent activation of the Met kinase domain and downstream MAPK and PI3K signaling pathways. This Aim will validate Met-ECD mutants as receptor antagonists in a variety of cell types and motivate future research into their therapeutic potential. Project Narrative The availability of functional recombinant Met-ECD will profoundly impact future research into mechanisms of tumorigenesis, bacterial pathogenesis, and embryonic development, and will provide insight into how these processes can be manipulated. The fact that the Met-ECD itself could also be developed for applications in molecular imaging or cancer therapy is another example of this project's high impact.