Development of Brain MRI Contrast Agents

脑MRI造影剂的开发

• 批准号: 10708637
• 负责人: Alan Koretsky
• 金额: $ 166.38万
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10708637
• 关键词: 2019-nCoV; Affinity; Antibodies; Binding; Biological Assay; Blood; Blood - brain barrier anatomy; Body Temperature; Brain; Brain Diseases; COVID-19 detection; COVID-19 therapeutics; Collaborations; Colorado; Contrast Media; Coupled; Coupling; Detection; Development; Diagnosis; Diagnostic; Dose; Drug Kinetics; Engineering; Extracellular Domain; Family; Goals; Hamsters; Health Sciences; Heterozygote; Human; Hybrids; Image; Immunoglobulin Fragments; Immunoglobulin G; In Vitro; Individual; Injections; Intravenous; Investigation; Joints; Kinetics; Laboratories; Lead; Magnetic Resonance Imaging; Manuscripts; Methods; Modification; Molecular; Mus; National Institute of Neurological Disorders and Stroke; Nature; Nebulizer; Neurologic; Neurosciences; Neurotensin; Pathologic; Patients; Peptides; Peptidyl-Dipeptidase A; Pharmacodynamics; Pharmacologic Substance; Preparation; Property; Publications; Publishing; Pulmonary Pathology; Radiation exposure; Radiology Specialty; Reagent; Regenerative Medicine; Reporting; Research Personnel; Research Project Grants; Resolution; SARS-CoV-2 infection; SARS-CoV-2 inhibitor; Scanning; Series; Signal Transduction; Supervision; TFRC gene; Testing; Text; Therapeutic; Time; Transgenic Mice; United States National Institutes of Health; Universities; University Health Services; Visualization; Washington; Work; antibody conjugate; blood-brain barrier function; brain magnetic resonance imaging; commercialization; cost; design; detection method; drug discovery; efficacy validation; experimental study; healthy volunteer; improved; in vivo; intravenous injection; iron oxide nanoparticle; mathematical model; molecular modeling; molecular pathology; mutant; nanobodies; nanoparticle; nervous system disorder; novel; novel therapeutics; pharmacokinetic model; receptor binding; sensor; tau Proteins; therapeutic candidate; transcytosis

We have made substantial progress towards Aims 1-5, 7&8. Specifically: a) We have reproducibly fabricated 3 nm iron oxide nanoparticles independently in our own lab. These serve as good T1 MRI contrast agents and have strong T2 contrast properties as well. b) We have developed several camelid VHH antibody fragment targeting domains for pathological targets relevant to neurological disease, and made specific modifications to enhance the affinity of the targeting domains. Several additional targeting domains are under development, including nanobodies for pathological relevant forms of the tau protein. We are also exploring the use of single chain antibodies derived from IgGs, which are larger than nanobodies but still substantially smaller than full sized IgGs. c) We have validated the efficacy of several blood-brain barrier transcytosis functionalization domains for mouse and human work. We have demonstrated blood brain barrier transcytosis in vivo by coupling these domains to the neurotensin peptide. When neurotensin crosses the blood brain barrier, it causes a rapid and transient reduction in body temperature, whereas neurotensin that remains in the blood does not have this effect. We have found robust and dose-dependent reduction in body temperature in mice injected intravenously with transferrin receptor binding nanobodies coupled to neurotensin, but no effect of the same concentrations of mutant nanobodies in which transferrin receptor binding is eliminated coupled to neurotensin. d) We have produced a novel transgenic mouse that expresses the extracellular domain of the human transferrin receptor in place of the homologous mouse domain to test the human specific nanobody that will be used for human studies. The mice are healthy as heterozygotes and appear to have normal blood brain barrier function at baseline. A nanobody that binds specifically to human transferrin receptor extracellular domain but does not bind mouse transferrin receptor appears to cross the blood brain barrier in these transgenic mice. e) We have determined that MP2RAGE MRI sequences have excellent properties for detecting T1 MRI contrast agents in both mice (at 4.7T and 9.4T) and humans (at 3T). Specifically, the test-retest reliability for MP2RAGE MRI was found to be superior to other T1 methods given similar scan times. One manuscript has been published (Sensitive detection of extremely small iron oxide nanoparticles in living mice using MP2RAGE with advanced image co-registration Scientific Reports (nature.com) and another manuscript has been submitted for publication. We are able to reliably detect as little as 0.04 mM iron oxide nanoparticle contrast agents f) We have optimized method for R2 and R2* MRI mapping with very high test retest reliability at 3T and 9.4T. We have tested these methods in healthy volunteers and in mice and found that they provide acceptable contrast, but that R1 mapping using MP2RAGE is superior with regard to sensitivity. A manuscript detailing these results has been submitted. g) We have performed extensive in vivo experiments in mice to assess molecular contrast agent kinetics using infrared tracking. We have constructed detailed mathematical models of the pharmacokinetics in living mice for contrast agents of various sizes. A manuscript has been published. IJMS Free Full-Text Pharmacokinetics of Single Domain Antibodies and Conjugated Nanoparticles Using a Hybrid near Infrared Method (mdpi.com) i) We developed a series of SARS-CoV-2 nanobodies that have potential for use as therapeutics, diagnostics, and laboratory reagents. A manuscript has been published. High affinity nanobodies block SARS-CoV-2 spike receptor binding domain interaction with human angiotensin converting enzyme Scientific Reports (nature.com). We have initiated three important collaborations: 1) With Dr. Richard Bowen at Colorado State University. Dr. Bowens lab demonstrated that nebulized treatment of hamsters with our lead nanobody protected them from SARS-CoV-2 infection and reduced lung pathology at 3 and 7 days after injection. An additional manuscript is in preparation. 2) With Dr. Terry Rabbitts at Quadrucepts Inc. Dr. Rabbitts group has engineered a tetravalent version of the nanobody with 10 fold higher affinity for SARS-CoV-2. We are currently testing this engineered nanobody. 3) With Dr. John Cirrito at Washington University in St Louis. Dr. Cirrito is using the nanobody to develop ultrasensitive electrochemical sensors for environmental detection of SARS-CoV-2. We are searching for additional partners for additional development and potential commercialization.

我们已经在目标1-5、7和8方面取得了重大进展。具体来说： a）我们在我们自己的实验室中独立地独立地制造了3 nm氧化铁纳米颗粒。它们是良好的T1 MRI对比剂，并且具有强大的T2对比特性。 b）我们为与神经系统疾病有关的病理靶标开发了几种Camelid VHH抗体片段靶向域，并进行了特定的修饰以增强靶向域的亲和力。正在开发几个其他靶向结构域，包括用于病理学相关形式的Tau蛋白的纳米体。我们还正在探索使用来自IgG的单链抗体的使用，该抗体大于纳米体，但仍然大于全尺寸的IgG。 c）我们已经验证了小鼠和人类工作的几个血脑屏障式脑介动函数化域的疗效。我们通过将这些结构域与神经素肽耦合通过偶联，在体内表现出了血脑屏障的转移。当神经素蛋白越过血脑屏障时，它会导致体温的快速和短暂降低，而保持血液中的神经素蛋白没有这种作用。我们发现，与神经辛蛋白偶联的转移蛋白受体结合纳米词的静脉内注射的小鼠中体温的稳健和剂量依赖性降低，但消除了相同浓度的突变纳米型的影响，在这些突变纳米型中，转移蛋白受体受体结合的相同浓度的影响被消除了与神经蛋白的结合。 d）我们产生了一种新型的转基因小鼠，该小鼠表达人类转铁蛋白受体的细胞外结构域代替同源小鼠结构域，以测试将用于人类研究的人类特异性纳米病。小鼠作为杂合子健康，在基线时似乎具有正常的血脑屏障功能。在这些转基因小鼠中，与人体转移蛋白受体外域但不结合小鼠转移蛋白受体似乎越过血液脑屏障的纳米病毒，但不结合小鼠转移蛋白受体。 e）我们已经确定MP2RAGE MRI序列具有出色的特性，可在小鼠（4.7 t和9.4t）和人类（3T）中检测T1 MRI对比剂。具体而言，发现MP2RAGE MRI的重新测试可靠性在给定相似的扫描时间的情况下优于其他T1方法。一份手稿已发表（使用具有先进图像共同注册的科学报告（Nature.com）的MP2RAGE对活小鼠中极小的氧化铁纳米颗粒的敏感检测（nature.com）和另一份手稿已提交出版。我们能够可靠地检测到尽可能少的检测。 0.04毫米氧化铁纳米颗粒对比剂 f）我们对R2和R2* MRI映射进行了优化的方法，在3T和9.4T时具有很高的测试可靠性。我们已经在健康的志愿者和小鼠中测试了这些方法，发现它们提供了可接受的对比度，但是使用MP2RAGE的R1映射在灵敏度方面是优越的。详细介绍了这些结果的手稿已提交。 g）我们在小鼠中进行了广泛的体内实验，以评估红外跟踪的分子对比剂动力学。我们已经为活小鼠中的药代动力学的详细数学模型构建了各种大小的对比剂。手稿已出版。 IJMS使用近近红外方法（MDPI.com）的单域抗体和共轭纳米颗粒的免费全文本药代动力学（MDPI.com） i）我们开发了一系列SARS-COV-2纳米型，它们具有用作治疗剂，诊断和实验室试剂的潜力。手稿已出版。高亲和力纳米体阻断SARS-COV-2尖峰受体结合结构域与人血管紧张素转化酶科学报告（Nature.com）。我们开始了三个重要的合作：1）与科罗拉多州立大学的理查德·鲍恩博士。 Bowens Lab博士证明，通过我们的铅纳米病毒对仓鼠的治疗保护了仓鼠免受SARS-COV-2感染的影响，并在注射后3和7天降低了肺病理。正在准备其他手稿。 2）与Quadrucepts Inc.的Terry Rabbitts博士一起，Rabbitts Group博士已经设计了四位纳米的四位化版本，对SARS-COV-2的亲和力高10倍。我们目前正在测试该工程的纳米机构。 3）与圣路易斯华盛顿大学的John Cirrito博士一起。 Cirrito博士正在使用纳米病毒来开发超敏感的电化学传感器，用于SARS-COV-2的环境检测。我们正在寻找其他合作伙伴以进行其他开发和潜在的商业化。