Development of Brain MRI Contrast Agents

我们在目标 1-5、7 和 8 方面取得了实质性进展。具体来说： a) 我们已经证明，具有强 pH 依赖性解除结合的突变型小鼠转铁蛋白受体结合纳米抗体在体内穿过 BBB 的效率比对照纳米抗体高约 500 倍。 b) 我们已经证明突变型小鼠转铁蛋白受体结合纳米抗体可以携带其他纳米抗体作为货物穿过小鼠血脑屏障。与 P2X7 受体结合的纳米抗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 BBB 运输的货物，并在其目标位点进行组织化学检测。 d) 我们已经证明，BBB 穿过纳米抗体的剂量反应曲线存在很强的非线性。静脉注射剂量大于 30 nmol/kg 时，脑内不会出现更多的物质积聚。达到的最大脑浓度为 4.5 nM。对于氧化铁纳米粒子的 MRI 检测来说，这个浓度太低了一个数量级。因此，我们决定转向 PET 成像以进一步开发分子对比成像剂。 e) 我们正在开发将纳米抗体与发射正电子的 Copper-64 结合以进行 PET 扫描的方法。 f) 我们与 Dale Kiesewetter 博士及其研究小组建立了新的合作关系，在注射后的多个时间点对活体小鼠进行 PET 扫描。 g) 我们已经与马里兰大学的 M Janoswki、乌普萨拉大学的 D Sehlin 和 S. Syvanen 以及 Ionis Pharmaceuticals 的 I Huggins 启动了新的合作，以使用我们的 BBB 交叉纳米体构建体进行额外的成像研究。 h) 我们与 USUHS 的 F. Nugent 博士、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，以测试我们的纳米抗体构建体在 TBI、中风和创伤后头痛小鼠模型中作为 BBB 穿越 P2X7 受体阻断疗法的功效。 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 纳米抗体有潜力用于气溶胶的实时环境监测和检测呼出气中的 SARS-CoV-2。

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.