MR Signal Amplification for Receptor Imaging

描述（由申请人提供）：将离体基因组和蛋白质组学筛选的最新成果转化为生命系统中标记物的可视化的迫切需要需要开发新的分子生物标记物成像技术。将分子成像应用于体内新标志物的能力将对疾病的早期检测、评估患者特异性治疗和监测疾病进展期间表达模式的动态变化具有重要意义。该应用建立在我们最近在设计、测试和应用酶介导的MR信号放大策略（MRamp）以成像分子靶点方面的创新之上。建议的研究是基于我们的观察，顺磁性酚在氧化还原酶的存在下，得到显着增强弛豫和MR信号。我们先前提出利用MRamp效应并将其应用于MR分子成像的需要。因此，我们完成了我们的研究从体外水平到体内实验的过渡。实现以下里程碑有助于实现研究目标：1）设计和放大合成MRamp底物; 2）确定与顺磁性部分连接的芳香族还原基团的性质决定底物特异性和酶选择性; 3）提供证据证明MRamp机制包括顺磁性寡聚体与大分子的聚合和结合; 4）优化抗受体抗体和扩增酶的小缀合物的完整方案（二元扩增系统）; 5）通过在EGFR表达肿瘤中使用MRI来执行所开发系统的测试; 6）测试替代扩增酶并鉴定用于未来研究的候选物。在上述主要研究结果的基础上，我们建议实现以下具体目标：具体目标。1.体内单酶靶向扩增成像系统的优化与测试。我们假设，MR信号放大策略可以通过优化体内递送来改善。这可以通过使用与EGFR抗体的F（ab '）2片段共价连接的单一酚氧化酶来实现。这一假设将在EGFR过表达肿瘤模型中进行检验。具体目标2。开发并测试双酶、双特异性方法，用于对共表达两种不同分子标志物的肿瘤细胞进行成像。我们假设，通过使用针对相同细胞上的不同靶点的两种抗体片段，我们将对粘附分子（EpCAM）和EGF受体在可能对联合抗体治疗有反应的非小细胞肺癌（NSCLC）细胞上的共表达进行成像。将在NSCLC脑转移模型中检验该假设。具体目标3。应用MRamp成像技术对体内肿瘤的炎症反应和受体库进行成像。我们假设，单核细胞/中性粒细胞成分的炎症反应，可以成像在肿瘤中分别从受体通过使用两个MRamp基板：第一个具有窄髓过氧化物酶特异性，第二个具有漆酶特异性。这一假设将在前列腺癌的转基因模型中得到验证。公共卫生相关性声明：开发能够有效消除肿瘤细胞或减缓心脏病和糖尿病的新药需要在实验室动物中进行充分的测试，以证明安全性和有效性。在活体动物中使用高精度检测这些细胞过程的医学扫描仪有可能显着缩短新药发现和后续临床使用之间的时间。这种减少的时间对患者和纳税人都有好处。我们提出的研究方法将导致开发新的工具（成像药物和组合物），用于医疗扫描仪。这些工具将用于跟踪与异常细胞相关的分子。这项研究将帮助科学家和医生检测这些细胞，并跟踪它们对药物的反应。

DESCRIPTION (provided by applicant): The pressing needs in translating recent achievements of genomic and proteomic screens ex vivo into the visualization of markers in living systems necessitate the development of novel molecular biomarker imaging techniques. The ability to apply molecular imaging to novel markers in vivo would have significant implications for early detection of disease, assessing patient-specific therapies and monitoring dynamic changes in expression patterns during disease progression. This application builds on our recent innovations in designing, testing and applying enzyme-mediated MR signal amplification strategy (MRamp) for imaging molecular targets. The proposed research is based on our observation that paramagnetic phenols in the presence of oxidoreductases give markedly enhanced relaxivity and MR signal. We previously proposed to harness MRamp effect and apply it for the needs of MR molecular imaging. As a result, we accomplished a transition of our research from in vitro level to in vivo experiments. Reaching the following milestones were instrumental in achieving the aims of the research: 1) designing and scaling up synthesis of MRamp substrates; 2) determining that the nature of aromatic reducing group linked to paramagnetic moiety defines substrate specificity and enzyme selectivity; 3) providing evidence that MRamp mechanism includes both polymerization and binding of paramagnetic oligomers to macromolecules; 4) optimizing a complete protocol for small conjugates of anti-receptor antibody and amplification enzymes ( binary amplification system ); 5) performing the testing of the developed system by using MRI in EGFR-expressing tumors; 6) testing alternative amplification enzymes and identifying a candidate for future research. By building on the above key findings we propose to achieve the following specific aims: Specific Aim. 1. Optimize and test in vivo single-enzyme targeted amplification imaging system. We hypothesize that MR signal amplification strategy could be improved by optimizing in vivo delivery. This can be achieved by using a) single, phenol- oxidizing enzyme covalently linked to F(ab')2 fragment of EGFR antibody. This hypothesis will be tested in EGFR-overexpressing tumor models. Specific Aim 2. Develop and test two-enzyme, bi-specific approach for imaging tumor cells co-expressing two different molecular markers. We hypothesize that by using two antibody fragments directed against different targets on the same cells we will image co-expression of adhesion molecule (EpCAM) and EGF receptor on non-small cell lung cancer (NSCLC) cells that are likely to respond to combined antibody therapy. This hypothesis will be tested in a model of NSCLC metastasis to the brain. Specific Aim 3. To use MRamp strategy for imaging inflammatory response and receptor repertoire of tumors in vivo. We hypothesize that monocyte/neutrophil component of inflammatory response can be imaged in tumors separately from receptors by using two MRamp substrates: the first having narrow myeloperoxidase specificity, and the second having laccase specificity. This hypothesis will be tested in transgenic model of prostate cancer. Public health relevance statement: The development of new drugs that can efficiently eliminate tumor cells or slow heart disease and diabetes requires ample testing in laboratory animals to prove safety and efficacy. The use of medical scanners that detect these cellular processes with high accuracy in live animals has the potential to significantly decrease the time between discovery of and subsequent clinical use of new medicines. This decreased time benefits both patients and taxpayers. We are proposing research approaches that will lead to the development of new tools (imaging drugs and compositions) for use with medical scanners. These tools will have applications for tracking the molecules that are linked to the abnormal cells. This research will help scientists and physicians to detect these cells and follow their response to medicines.