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

用于 Met 靶向癌症成像和治疗的工程化 HGF-NK1 拮抗剂

• 批准号: 8100279
• 负责人: JENNIFER R COCHRAN
• 金额: $ 32.06万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8100279
• 关键词: 2-Fluoro-2-deoxyglucose; Affinity; Alanine; Animal Model; Apoptosis; Area; Binding; Biochemical; Biodistribution; Biological; Biological Assay; Bioluminescence; Blood capillaries; CD34 gene; Cancer Biology; Cancer Diagnostics; Cancer Etiology; Cell Culture Techniques; Cell Proliferation; Cell surface; Circular Dichroism; Clinical; Clinical Trials; Complement; Cysteine; Development; Dimerization; Disease Management; Drug Design; Drug Kinetics; Engineering; Exhibits; FDA approved; Fluorescent Dyes; Funding; Generations; Goals; Growth; Heating; Heparin; Hepatocyte Growth Factor; Homodimerization; Hot Spot; Human; Image; Imagery; Immunohistochemistry; In Vitro; Individual; Kringles; Label; Life; Ligands; Location; Luciferases; Lysine; Malignant Neoplasms; Mammalian Cell; Mammary Neoplasms; Measures; Mediating; Metabolism; Modeling; Molecular Sieve Chromatography; Monitor; Mus; Mutagenesis; Mutation; Mutation Analysis; N-terminal; Neoplasm Metastasis; Optics; Patients; Phase I Clinical Trials; Point Mutation; Positron-Emission Tomography; Pre-Clinical Model; Primary Neoplasm; Property; Prostate carcinoma; Proteins; Radioisotopes; Radiolabeled; Receptor Activation; Receptor Protein-Tyrosine Kinases; Recombinants; Research; Sampling; Scanning; Signal Pathway; Signal Transduction; Site; Staging; Staining method; Stains; Structure; Structure-Activity Relationship; Surface Plasmon Resonance; System; Temperature; Testing; Therapeutic; Therapeutic Agents; Therapeutic Intervention; Tissues; Translations; Treatment Efficacy; Tube; Tumor Cell Line; United States National Institutes of Health; Work; Xenograft Model; Xenograft procedure; amino group; angiogenesis; base; cancer diagnosis; cancer imaging; cancer therapy; capillary; chelation; density; design; directed evolution; flexibility; gain of function; high throughput screening; imaging probe; improved; in vivo; inhibitor/antagonist; insight; light scattering; loss of function; lung Carcinoma; meetings; migration; molecular imaging; mutant; neoplastic cell; novel strategies; optical imaging; outcome forecast; pre-clinical; preclinical study; public health relevance; radiotracer; receptor; receptor binding; receptor expression; research study; response; therapeutic target; tumor; tumor growth; tumor progression; tumor xenograft; uptake; von Willebrand Factor

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: We developed high affinity, protein-based inhibitors of Met, a receptor that is over-expressed on many human cancers. These engineered Met-targeting proteins will be used to open up new research areas in cancer biology, cancer therapy, molecular imaging, and structure-based drug design. The studies we are proposing will also evaluate the potential of these engineered Met-targeting proteins for clinical translation as cancer diagnostics and therapeutics.

描述（由申请人提供）：介导增殖，存活和迁移的细胞信号通路失调是癌症的根本原因，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表达与许多人类肿瘤的预后不良相关，使其成为治疗干预的有吸引力的靶标。目前尚无针对MET受体的FDA批准的治疗剂。但是，一些候选分子最近进入了早期临床试验。因此，有效抑制MET受体激活的分子将对癌症治疗产生重大临床影响。此外，开发针对MET靶向的分子成像剂的研究非常有限。此类成像剂的可用性将有助于癌症诊断，分期和疾病管理，并有助于确定将成为符合靶向疗法的良好候选者的患者。为了开发强大的征靶剂，我们使用了天然MET激活配体的N端子和第一克林格结构域（NK1），肝细胞生长因子（HGF）作为工程有效的MET受体拮抗剂的基础。使用定向进化，我们设计了NK1突变体，与野生型NK1相比，MET结合亲和力和热稳定性有显着改善。引入这些NK1蛋白的理性设计的位置定向突变将它们转化为MET受体拮抗剂。 在该提案的AIM 1中，我们将对荧光标记和放射标记的NK1突变体进行临床前研究，以测试其在生命受试者中非侵入性图像MET表达的能力，目的是将其作为体内分子成像剂中的形式开发。在AIM 2中，我们将进行临床前研究，以测量几种小鼠肿瘤模型中NK1突变体对肿瘤生长，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原发性肿瘤和转移的生长和进展，并监测肿瘤部位的MET表达和代谢的变化。在AIM 3中，我们将充分表征较大的工程NK1突变体与表达MET肿瘤细胞的结合，并将确定其二聚体和随后抑制MET受体激活的能力。在AIM 4中，我们将使用这些工程的NK1蛋白来探测MET受体系统中配体受体相互作用的序列结构功能关系，并为其作用机理提供生化和生物物理学的见解。在所有四个目标中，将将结果与野生型NK1进行比较，以确定MET受体结合亲和力和蛋白质稳定性对细胞培养和动物模型中生物学活性的影响。该提案完成后，我们将评估工程NK1蛋白作为分子成像和治疗剂在临床前模型中的潜力，这是临床翻译道路的重要一步。 公共卫生相关性：我们开发了高亲和力，基于蛋白质的MET抑制剂，MET是一种对许多人类癌症过表达的受体。这些设计的靶向蛋白质将用于打开癌症生物学，癌症治疗，分子成像和基于结构的药物设计方面的新研究领域。我们提出的研究还将评估这些工程征收的靶向蛋白作为临床翻译作为癌症诊断和治疗剂的潜力。