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.

我们在实现目标1-5、7和8方面取得了重大进展。具体而言： a）我们已经在我们自己的实验室中独立地可重复地制造了3 nm氧化铁纳米颗粒。它们作为良好的T1 MRI造影剂，也具有较强的T2造影特性。 B）我们已经开发了几种骆驼VHH抗体片段靶向结构域，用于与神经疾病相关的病理靶标，并进行了特异性修饰以增强靶向结构域的亲和力。几个额外的靶向结构域正在开发中，包括用于病理相关形式的tau蛋白的纳米抗体。我们还在探索使用来自IgG的单链抗体，其比纳米抗体大，但仍比全尺寸IgG小得多。 c）我们已经验证了几种血脑屏障转胞吞功能化结构域对于小鼠和人类工作的功效。我们已经通过将这些结构域与神经降压素肽偶联来证明体内血脑屏障转胞吞作用。当神经降压素穿过血脑屏障时，它会导致体温迅速和短暂的降低，而留在血液中的神经降压素则没有这种效果。我们已经发现，在静脉内注射了与神经降压素偶联的转铁蛋白受体结合纳米抗体的小鼠中，体温出现了稳健的和剂量依赖性的降低，但其中转铁蛋白受体结合被消除与神经降压素偶联的相同浓度的突变纳米抗体没有影响。 d）我们已经产生了一种新的转基因小鼠，其表达人转铁蛋白受体的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抗体。小鼠作为杂合子是健康的，并且在基线时似乎具有正常的血脑屏障功能。特异性结合人转铁蛋白受体胞外结构域但不结合小鼠转铁蛋白受体的纳米抗体似乎在这些转基因小鼠中穿过血脑屏障。 e）我们已经确定，MP2 γ MRI序列对于检测小鼠（在4.7T和9.4T下）和人类（在3 T下）中的T1 MRI造影剂具有优异的特性。具体而言，MP2 MRI的重测信度优于其他T1方法（扫描时间相似）的上级信度。已经发表了一份手稿（使用MP2生物传感器在活体小鼠中灵敏检测极小的氧化铁纳米颗粒，具有先进的图像配准科学报告（nature.com）），另一份手稿已提交出版。我们能够可靠地检测低至0.04 mM的氧化铁纳米颗粒造影剂 f）我们优化了R2和R2* MRI标测的方法，在3 T和9.4T下具有非常高的重测信度。我们已经在健康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MP 2 β的R1映射在灵敏度方面是上级的。已经提交了一份详细说明这些结果的手稿。 g）我们已经在小鼠中进行了广泛的体内实验，以使用红外跟踪评估分子造影剂动力学。我们已经建立了详细的数学模型，在活体小鼠的药物动力学的各种大小的造影剂。手稿已经出版。使用混合近红外方法的单结构域抗体和结合纳米颗粒的IJMS免费全文药代动力学（mdpi.com） i）我们开发了一系列SARS-CoV-2纳米抗体，这些纳米抗体具有用作治疗剂、诊断剂和实验室试剂的潜力。手稿已经出版。高亲和力纳米抗体阻断SARS-CoV-2刺突受体结合域与人血管紧张素转换酶的相互作用科学报告（nature.com）。我们已经启动了三个重要的合作：1）与科罗拉多州立大学的理查德·鲍恩博士。Bowens博士的实验室证明，用我们的铅纳米抗体对仓鼠进行雾化治疗可以保护它们免受SARS-CoV-2感染，并在注射后3天和7天减少肺部病理学。另一份手稿正在编写中。 2)与Quadrucepts Inc.的Terry Rabbitts博士合作。Rabbitts博士的团队已经设计了一种四价的纳米抗体，对SARS-CoV-2的亲和力高出10倍。我们目前正在测试这种工程纳米抗体。3)与圣刘易斯的华盛顿大学的约翰·西里托博士一起。Cirrito博士正在使用纳米抗体开发用于SARS-CoV-2环境检测的超灵敏电化学传感器。我们正在寻找更多的合作伙伴，以进行进一步的开发和潜在的商业化。