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方面取得了实质性进展。 A)我们在自己的实验室中独立地重复制造了3纳米氧化铁纳米颗粒。它们既是良好的T1磁共振造影剂，又具有很强的T2对比度特性。 B)针对与神经系统疾病相关的病理靶点，我们开发了几个驼状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。其他几个靶向域正在开发中，包括病理相关形式的tau蛋白的纳米抗体。我们还在探索使用来自免疫球蛋白的单链抗体，这种抗体比纳米抗体大，但仍然比全尺寸免疫球蛋白小得多。 C)我们已经验证了几个血脑屏障跨细胞功能化结构域在小鼠和人类工作中的有效性。我们已经通过将这些结构域与神经降压素多肽偶联，在体内证明了血脑屏障跨细胞作用。当神经降压素穿过血脑屏障时，它会导致体温迅速而短暂的下降，而留在血液中的神经降压素则没有这种作用。我们发现，静脉注射与神经降压素偶联的转铁蛋白受体结合纳米体小鼠的体温显著降低，且呈剂量依赖性，但与神经降压素偶联的相同浓度的转铁蛋白受体结合被消除的突变纳米体对小鼠的体温没有影响。 D)我们制造了一种新型的转基因小鼠，它表达人转铁蛋白受体的胞外域，而不是同源的小鼠域，以测试将用于人类研究的人类特定纳米体。这些小鼠是健康的杂合子，在基线水平上似乎具有正常的血脑屏障功能。在这些转基因小鼠中，一种与人转铁蛋白受体胞外区域特异结合但不与小鼠转铁蛋白受体结合的纳米体似乎跨越了血脑屏障。 E)我们已经确定，MP2RAGE MRI序列对于检测小鼠(4.7T和9.4T)和人类(3T)的T1磁共振造影剂具有极好的性能。具体地说，MP2RAGE MRI的重测可靠性被发现在扫描时间相似的情况下优于其他T1方法。一份手稿已经发表(使用MP2RAGE和先进的图像联合注册科学报告(Nature.com)在活体小鼠中灵敏地检测到极小的氧化铁纳米颗粒)，另一份手稿已提交出版。我们能够可靠地检测出低至0.04 mm的纳米氧化铁造影剂 F)我们优化了R2和R2*MRI定位的方法，在3T和9.4T下具有很高的重测可靠性。我们在健康志愿者和小鼠身上测试了这些方法，发现它们提供了可以接受的对比度，但使用MP2RAGE的R1映射在灵敏度方面更好。已经提交了详细说明这些结果的手稿。 G)我们已经在小鼠身上进行了广泛的体内实验，以使用红外跟踪来评估分子造影剂的动力学。我们已经建立了不同大小造影剂在活体小鼠体内药代动力学的详细数学模型。已经出版了一份手稿。用混合近红外法研究单域抗体和结合纳米粒的IJMS免费全文药代动力学(mdpi.com) I)我们开发了一系列SARS-CoV-2纳米体，这些纳米体具有作为治疗、诊断和实验室试剂的潜力。已经出版了一份手稿。高亲和力纳米抗体阻断SARS-CoV-2刺激性受体结合域与人类血管紧张素转换酶科学报告(Nature.com)的相互作用。我们启动了三项重要的合作：1)与科罗拉多州立大学的理查德·鲍文博士合作。鲍文斯博士的实验室证明，用我们的铅纳米体雾化处理仓鼠，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。另一份手稿正在准备中。2)与Quadrucepts Inc.的Terry Rabbitts博士合作，Rabbitts博士团队设计了一种对SARS-CoV-2亲和力高10倍的四价体纳米体。我们目前正在测试这种经过工程处理的纳米物体。3)与圣路易斯华盛顿大学的约翰·西里托博士。Cirrito博士正在利用这种纳米体开发超灵敏的电化学传感器，用于检测SARS-CoV-2的环境。我们正在寻找更多的合作伙伴进行进一步的开发和潜在的商业化。