Development of Brain MRI Contrast Agents

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）我们已经证明，具有强pH依赖性未结合的突变小鼠转铁蛋白受体结合纳米抗体在体内比对照纳米抗体更有效地穿过BBB约500倍。 B）我们已经证明突变的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BB B。与P2 X7受体结合的纳米抗体的串联二聚体已被用作模型货物。 c）我们已经表明，在注射后1至16小时之间，可以在毛细血管耗尽的脑裂解物中检测到转运穿过BBB的货物，并在其靶位点进行组织化学检测。 d）我们已经证明，在BBB交叉纳米抗体的剂量-反应曲线中存在强非线性。在静脉注射的剂量大于30 nmol/kg时，没有更多的货物脑蓄积。达到的最大脑浓度为4.5 nM。该浓度对于氧化铁纳米颗粒的MRI检测来说太低了一个数量级。因此，我们决定转向PET成像，以进一步开发分子造影剂。 e）我们正在开发将纳米抗体缀合到正电子发射铜-64用于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）我们已经与马里兰州的M Janoswki、D Sehlin和S.乌普萨拉大学的Syvanen和Ionis Pharmaceuticals的I Huggins使用我们的BBB交叉纳米抗体构建体进行额外的成像研究。 我们与F博士建立了新的合作关系。USUHS的Nugent，N博士。慕尼黑大学的普莱斯尼拉和F. Porreca在U Arizona测试我们的纳米抗体构建体作为BBB交叉P2 X7受体阻断疗法在TBI、中风和创伤后头痛的小鼠模型中的功效。 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真实的环境监测和呼出气中的SARS-CoV-2检测。

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.