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传导途径的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。特别地，Met酪氨酸激酶受体的失调和过度表达与许多人类肿瘤中的不良预后相关，使其成为治疗干预的有吸引力的靶标。目前还没有FDA批准的针对Met受体的治疗药物;然而，一些候选分子最近已进入早期临床试验阶段。因此，有效抑制Met受体活化的分子将对癌症治疗具有显著的临床影响。此外，开发用于体内Met表达的非侵入性可视化的Met靶向分子成像剂的研究极其有限。这种显像剂的可用性将有助于癌症诊断，分期和疾病管理，以及帮助确定谁是Met靶向治疗的良好候选人的患者。为了开发稳健的Met靶向剂，我们使用天然Met活化配体肝细胞生长因子（HGF）的N末端和第一Kringle结构域（NKl）作为工程化有效Met受体拮抗剂的基础。使用定向进化，我们设计了与野生型NK 1相比，在Met结合亲和力和热稳定性方面具有显著改善的NK 1突变体。通过将基因设计的定点突变引入这些NK 1蛋白质中，将其转化为Met受体拮抗剂。 在该提案的目标1中，我们将对荧光标记和放射性标记的NK 1突变体进行临床前研究，以测试它们在活体受试者中非侵入性成像Met表达的能力，目标是将它们开发为体内分子成像剂。在目标2中，我们将进行临床前研究，以测量NK 1突变体在几种小鼠肿瘤模型中对肿瘤生长、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瘤的生长和进展，并监测肿瘤部位Met表达和代谢的变化。在目标3中，我们将充分表征更大范围的工程NK 1突变体与表达Met的肿瘤细胞的结合，并确定它们二聚化并随后抑制Met受体激活的能力。在目标4中，我们将使用这些工程化的NK 1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，并提供对其作用机制的生物化学和生物物理学见解。在所有四个目标中，将结果与野生型NK 1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物和动物模型中生物活性的影响。在完成这项提案后，我们将评估工程化NK 1蛋白作为临床前模型中分子成像和治疗剂的潜力，这是临床转化道路上的重要一步。 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是一种在许多人类癌症中过度表达的受体。这些工程化的Met靶向蛋白将用于开辟癌症生物学、癌症治疗、分子成像和基于结构的药物设计的新研究领域。我们提出的研究还将评估这些工程Met靶向蛋白作为癌症诊断和治疗的临床翻译的潜力。