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

描述（由申请人提供）：Met受体酪氨酸激酶对介导细胞增殖、存活和迁移至关重要。人类肿瘤的侵袭和转移倾向与Met的失调有关；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细胞外结构域（Met- ecd）是Met活化的有效拮抗剂。不幸的是，目前重组表达Met-ECD的方法产量低，阻碍了其生物学特性的全面表征和作为潜在治疗方法的部署。我们的实验室拥有强大且成熟的技术来进化、优化和表达这些具有挑战性的蛋白质，Met-ECD是应用这项技术的理想选择。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细胞外结构域，使其在酵母中高可溶性表达。利用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：1)具有天然受体折叠，2)在酵母中具有高可溶性表达水平（mg/L）。目的2：测定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Aim 1中分离的Met-ECD突变体，以验证具有改善表达的工程受体保留天然配体结合特性和生物学功能。我们将Met- ecd突变体融合到野生型Met跨膜和细胞内结构域，并将这些构建体转染到表达低水平内源性Met的哺乳动物细胞系中。我们将测试这些细胞系结合HGF配体和诱导细胞信号传导的能力，并将它们与转染野生型Met的细胞进行比较。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提供验证。目的3：确定可溶性Met-ECD突变体与肿瘤细胞结合并抑制信号传导的能力。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功能来确定它们的能力：1)结合表达Met的肿瘤细胞；2)抑制配体依赖性和非配体依赖性的Met激酶结构域激活以及下游MAPK和PI3K信号通路。这一目的将验证Met-ECD突变体在各种细胞类型中的受体拮抗剂作用，并激发对其治疗潜力的未来研究。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见解。Met-ECD本身也可以开发用于分子成像或癌症治疗的应用，这是该项目高影响力的另一个例子。

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.