Engineered HGF-NK1 antagonists for Met-targeted cancer imaging and therapy

项目摘要/摘要 调节细胞增殖、存活和迁移的信号通路失调是一种 癌症的根本原因，并导致许多人类肿瘤的侵袭和转移。特别是， Met酪氨酸激酶受体的失调和过度表达与许多患者的预后不良有关 人类肿瘤，使其成为治疗干预的有吸引力的靶点。目前没有FDA批准的 靶向Met受体的治疗；然而，一些候选分子最近进入了早期阶段 临床试验。因此，有效抑制蛋氨酸受体激活的分子将具有重要的临床意义 对癌症治疗的影响。此外，开发甲硫氨酸靶向分子显像剂的研究 体内甲硫氨酸表达的侵袭性可视化极其有限。这种成像的可用性 代理人将有助于癌症诊断、分期和疾病管理，并帮助识别 将是Met靶向治疗的很好的候选者。为了开发强大的Met靶向试剂，我们使用了N- 天然蛋氨酸激活配体肝细胞生长因子的末端和第一个Kringle结构域(NK1)，AS 为设计有效的Met受体拮抗剂奠定了基础。利用定向进化，我们设计了NK1突变体 与野生型NK1相比，在蛋氨酸结合亲和力和热稳定性方面有显著改善。 将合理设计的定点突变引入这些NK1蛋白中，将它们转化为蛋氨酸 受体拮抗剂。 在建议的目标1中，我们将对荧光标记和放射性标记的NK1进行临床前研究 突变体测试他们在活着的受试者中非侵入性成像Met表达的能力，目标是 开发它们作为体内分子显像剂。在目标2中，我们将进行临床前研究，以测量 NK1突变体对几种小鼠肿瘤模型中肿瘤生长、转移和血管生成的影响。 在治疗过程中，将使用非侵入性成像来监测肿瘤的生长和进展 监测原发肿瘤和转移的情况，并监测肿瘤部位Met表达和代谢的变化。在……里面 目的3，我们将充分表征一组更大的工程NK1突变体与Met表达的结合 并将确定其二聚体的能力，并随后抑制Met受体的激活。在AIM 4，我们将使用这些工程的NK1蛋白来探索配体的序列-结构-功能关系- Met受体系统中的受体相互作用，并提供对其生物化学和生物物理的洞察 作用机制。在所有四个目标中，结果将与野生型NK1进行比较，以确定Met的效果 细胞培养和动物模型中受体结合亲和力和蛋白质稳定性对生物活性的影响。vt.在.的基础上 完成这项计划后，我们将评估工程NK1蛋白作为分子的潜力 临床前模型中的成像和治疗剂，是临床转化道路上的重要一步。

Project Summary/Abstract Dysregulation of cell signaling pathways that mediate proliferation, survival, and migration are an underlying cause of cancer, and result in invasion and metastasis of many human tumors. In particular, dysregulation and over-expression of the Met tyrosine kinase receptor correlates to poor prognosis in many human tumors, making it an attractive target for therapeutic intervention. There are currently no FDA-approved therapeutics targeting the Met receptor; however, a few candidate molecules have recently entered early stage clinical trials. Therefore, molecules that potently inhibit Met receptor activation would have a significant clinical impact on cancer therapy. In addition, studies to develop Met-targeted molecular imaging agents for non- invasive visualization of Met expression in vivo have been extremely limited. The availability of such imaging agents would aid in cancer diagnosis, staging, and disease management, as well as help identify patients who would be good candidates for Met-targeted therapies. To develop robust Met-targeting agents we used the N- terminal and first Kringle domain (NK1) of the natural Met activating ligand, hepatocyte growth factor (HGF), as a basis for engineering potent Met receptor antagonists. Using directed evolution, we engineered NK1 mutants with significant improvements in Met binding affinity and thermal stability compared to wild-type NK1. Rationally-designed, site-directed mutations introduced into these NK1 proteins transformed them into Met receptor antagonists. In Aim 1 of the proposal, we will perform pre-clinical studies on fluorescently-labeled and radiolabeled NK1 mutants to test their ability to non-invasively image Met expression in living subjects, with the goal of developing them as in vivo molecular imaging agents. In Aim 2, we will perform pre-clinical studies to measure the effects of NK1 mutants on tumor growth, metastasis, and angiogenesis in several mouse tumor models. During the course of treatment, non-invasive imaging will be used to monitor growth and progression of the primary tumor and metastases, and to monitor changes in Met expression and metabolism at the tumor site. In Aim 3, we will fully characterize the binding of a larger panel of engineered NK1 mutants to Met-expressing tumor cells, and will determine their ability to dimerize and subsequently inhibit Met receptor activation. In Aim 4, we will use these engineered NK1 proteins to probe sequence-structure-function relationships of ligand- receptor interactions in the Met receptor system, and provide biochemical and biophysical insight into their mechanism of action. In all four aims, results will be compared to wild-type NK1 to determine the effects of Met receptor binding affinity and protein stability on biological activity in cell culture and animal models. Upon completion of this proposal, we will have evaluated the potential of engineered NK1 proteins as molecular imaging and therapeutic agents in pre-clinical models, an important step on the path to clinical translation.