Development of Brain MRI Contrast Agents

我们在实现目标1-5、7和8方面取得了实质性进展。 A)我们已经证明，突变的小鼠转铁蛋白受体结合纳米体具有强烈的pH依赖性解离，在体内跨越血脑屏障的效率大约是对照纳米体的500倍。 B)我们已经证明了突变的小鼠转铁蛋白受体结合纳米体可以携带其他纳米体作为货物穿过小鼠的血脑屏障。一种与P2X7受体结合的纳米体串联二聚体已被用作模型货物。 C)我们已经证明，在注射后1至16小时内，可以在毛细血管耗尽的脑裂解物中检测到跨血脑屏障运输的货物，并在其目标位置进行组织化学检测。 D)我们已经证明了BBB跨越纳米体的剂量-响应曲线具有很强的非线性。当静脉注射剂量大于30nmol/kg时，不会有更大的脑积聚货物。达到的最大脑浓度为4.5 NM。这一浓度对于MRI检测氧化铁纳米颗粒来说太低了一个数量级。因此，我们决定转向PET成像，以进一步开发分子对比显像剂。 E)我们正在开发将纳米实体与发射正电子的铜-偶联的方法，用于正电子发射断层扫描。 F)我们与Dale Kiesewetter博士和他的研究小组建立了一个新的合作关系，在注射后的多个时间点对活着的小鼠进行PET扫描。 G)我们与马里兰州大学的M Janoswki、Uppsala U的D Sehlin和S.Syvanen以及Ionis PharmPharmticals的I Huggins展开了新的合作，使用我们的BBB交叉纳米体结构进行额外的成像研究。 H)我们已经与USUHS的F.Nugent博士、德国慕尼黑大学的N.Plesnila博士和亚利桑那大学的F.Porreca博士建立了新的合作关系，以测试我们的纳米体构造在脑外伤、中风和创伤后头痛小鼠模型中作为血脑屏障跨P2X7受体阻断疗法的有效性。 I)我们在华盛顿大学的合作者正在进行的工作表明，我们的SARS-CoV-2纳米抗体具有用于实时环境监测气溶胶和检测呼气中SARS-CoV-2的潜力。

We have made substantial progress towards Aims 1-5, 7&8. Specifically: a) We have demonstrated that a mutant mouse transferrin receptor binding nanobody with strong pH dependent unbinding crosses the BBB in vivo approximately 500 times more effectively than a control nanobody. b) We have demonstrated that the mutant mouse transferrin receptor binding nanobody can carry other nanobodies as cargo across the mouse BBB. A tandem dimer of nanobodies that bind to the P2X7 receptor has been used as a model cargo. c) We have shown that cargo transported across the BBB can be detected in capillary depleted brain lysates and histochemically at their target sites between 1 and 16 hours after injection. d) We have demonstrated that there is a strong nonlinearity in the dose-response curve for the BBB crossing nanobodies. At doses greater than 30 nmol/kg injected intravenously, there is no greater brain accumulation of cargo. The maximum brain concentration achieved was 4.5 nM. This concentration is an order of magnitude too low for MRI detection of iron oxide nanoparticles. We therefore have decided to switch to PET imaging for further development molecular contrast imaging agents. e) We are developing methods to conjugate nanobodies to the positron emitting Copper-64 for PET scans. f) We have established a new collaboration with Dr. Dale Kiesewetter and his research group to perform PET scans in living mice at multiple time points after injection. g) We have initiated new collaborations with M Janoswki at U Maryland, D Sehlin and S. Syvanen at Uppsala U, and I Huggins at Ionis Pharmaceuticals to perform additional imaging studies using our BBB crossing nanobody constructs. h) We have established new collaborations with Dr F. Nugent at USUHS, Dr. N. Plesnila at U Munich, and Dr. F. Porreca at U Arizona to test our nanobody constructs for efficacy as BBB crossing P2X7 receptor blocking therapeutics in mouse models of TBI, stroke, and post-traumatic headache. i) Ongoing work by our collaborators at Washington University indicates that our SARS-CoV-2 nanobodies have potential for use in real time environmental surveillance of aerosols and for detection of SARS-CoV-2 in exhaled breath.