Pilot studies to develop probes for in vivo imaging of P13K/Akt pathway activity

开发 P13K/Akt 通路活性体内成像探针的试点研究

• 批准号: 7212974
• 负责人: Mark M Moasser
• 金额: $ 15.38万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-01-01 至 2008-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7212974
• 关键词: Affinity; Antibodies; Antineoplastic Agents; Apoptotic; Bacteriophages; Benchmarking; Binding; Biochemical; Biological Markers; Biological Models; Breast Cancer Cell; Cell Surface Proteins; Cell model; Cell surface; Cells; Clinical; Cultured Cells; Development; Dose; Doxycycline; Engineering; Epitopes; Evaluation; Event; Exploratory/Developmental Grant; Funding; Gene Expression; Human; Image; Imaging technology; Individual; Lead; Libraries; Link; Malignant Neoplasms; Measures; Membrane; Metabolic; Modality; Modeling; Monitor; Mus; PTEN gene; Pathway interactions; Patient Selection; Patients; Phase; Pilot Projects; Positioning Attribute; Proteome; Radiolabeled; Relapse; Reporter; Research Personnel; Resistance; Risk; Screening procedure; Signal Pathway; Signal Transduction; Specificity; Surface; System; Techniques; Technology; Tetanus Helper Peptide; Transmembrane Transport; Xenograft procedure; annexin A5; base; cell type; extracellular; feeding; imaging probe; in vivo; mouse model; neoplastic cell; novel strategies; programs; radiochemical; radiotracer; response; tool; trafficking; transcription factor; tumor; tumor xenograft; tumorigenesis

DESCRIPTION (provided by applicant): Akt sits at the crossroads of numerous signal tansduction pathways essential for human tumorigenesis and activation of the PI3K/Akt signaling is a hallmark of many human cancers. Inhibition of this critical transforming pathway is closely linked with the anti-tumor effects of many targeted therapies and the ability to inhibit Akt signaling in cell culture models has become an important benchmark for the evaluation of targeted anti-cancer drugs and a molecular marker of response and resistance to many therapies. The ability to non-invasively measure PI3K/Akt signaling activity in vivo is currently lacking. Such a modality could allow much better patient selection for therapies, could predict reponse to therapy and could potentially predict relapse on therapy. Such a modality would allow the unprecedented opportunity for dose escalation or reduction in individual patients guided by a readout of tumor PI3K/Akt activity. An imaging modality to monitor Akt activity in vivo is urgently needed. The technology to directly measure intracellular signaling events in tumors in vivo is currently not available. However cell surface probes can potentially be used as reporters of intraceilular signaling as evidenced by the development of annexin V as a probe of early intracellular apoptotic signaling. Indeed the PI3K/Akt signaling pathway regulates endosomal trafficking and membrane transport, including the expression of several membrane metabolic transporters, and regulates gene expression through several transcription factors and it is likely to modulate the cell surface proteome. Based on the hypothesis that tumor cells express certain surface epitopes uniquely in the PI3K/Akt activated state, we seek to develop probes that bind such epitopes and have developed a cell model well suited for this effort. Our cell model consists of engineered MDA-468 breast cancer cells which have highly active Akt signaling due to deletion of PTEN, and in which we can rapidly inactivate Akt signaling by tet-induced expression of PTEN. We propose to develop probes that will bind to the surface of these cells in the Akt-active state, but not the Akt-inactive state. We will subtractively screen a single chain antibody (scFv) phage library to identify probes that will bind selectively to the surface of Akt-activated cells, but not the Akt-inactive cells. Preliminarily identified probes will be further validated in broader cell contexts. Validated probes will be F-18 radiolabelled, and the sensitivity of candidate probes in generating a signal in Akt-activated tumors will be preliminarily assessed by micro-PET studies of mice bearing uninduced MDA-468TR-PTEN xenograft tumors. The specificity of such probes will be studied by microPET studies of the same mice after doxycycline feeding to inactive tumor Akt signaling. If successful, this modality could potentially lead to a ground-breaking predictive clinical monitoring tool.

描述（由申请人提供）：Akt 位于人类肿瘤发生所必需的众多信号传导途径的十字路口，PI3K/Akt 信号传导的激活是许多人类癌症的标志。抑制这一关键转化途径与许多靶向治疗的抗肿瘤作用密切相关，在细胞培养模型中抑制Akt信号传导的能力已成为评价靶向抗癌药物的重要基准和反应的分子标志物以及对多种疗法的耐药性。目前缺乏体内非侵入性测量 PI3K/Akt 信号传导活性的能力。这种方式可以让患者更好地选择治疗方法，可以预测对治疗的反应，并有可能预测治疗的复发。这种方式将为个体患者提供前所未有的机会，在肿瘤 PI3K/Akt 活性读数的指导下增加或减少剂量。迫切需要一种监测体内 Akt 活性的成像方式。目前还没有直接测量体内肿瘤细胞内信号传导事件的技术。然而，细胞表面探针有可能用作细胞内信号传导的报告基因，膜联蛋白 V 作为早期细胞内凋亡信号传导探针的发展证明了这一点。事实上，PI3K/Akt 信号通路调节内体运输和膜运输，包括几种膜代谢转运蛋白的表达，并通过多种转录因子调节基因表达，并且可能调节细胞表面蛋白质组。基于肿瘤细胞在 PI3K/Akt 激活状态下独特表达某些表面表位的假设，我们寻求开发结合此类表位的探针，并开发了非常适合这项工作的细胞模型。我们的细胞模型由工程化的 MDA-468 乳腺癌细胞组成，这些细胞由于 PTEN 的缺失而具有高度活跃的 Akt 信号传导，并且我们可以通过 tet 诱导的 PTEN 表达快速使 Akt 信号传导失活。我们建议开发能够与 Akt 活性状态（而非 Akt 非活性状态）的细胞表面结合的探针。我们将消减筛选单链抗体 (scFv) 噬菌体库，以鉴定可选择性结合 Akt 激活细胞表面但不结合 Akt 非活性细胞表面的探针。初步鉴定的探针将在更广泛的细胞环境中得到进一步验证。验证的探针将进行 F-18 放射性标记，候选探针在 Akt 激活的肿瘤中产生信号的敏感性将通过对携带未诱导的 MDA-468TR-PTEN 异种移植肿瘤的小鼠进行微型 PET 研究来初步评估。此类探针的特异性将通过对相同小鼠进行 microPET 研究来研究，这些小鼠在使用多西环素喂养非活性肿瘤 Akt 信号后。如果成功，这种方式可能会带来突破性的预测性临床监测工具。