Department of Public Health and Caring Sciences/Geriatrics, Uppsala University, Rudbeck Laboratory, 75185 Uppsala, Sweden.
Neuroimage. 2017 Mar 1;148:55-63. doi: 10.1016/j.neuroimage.2017.01.004. Epub 2017 Jan 6.
Antibodies are highly specific for their target molecules, but their poor brain penetrance has restricted their use as PET ligands for imaging of targets within the CNS. The aim of this study was to develop an antibody-based radioligand, using the Tribody format, for PET imaging of soluble amyloid-beta (Aβ) protofibrils, which are suggested to cause neurodegeneration in Alzheimer's disease. Antibodies, even when expressed in smaller engineered formats, are large molecules that do not enter the brain in sufficient amounts for imaging purposes. Hence, their transport across the blood-brain barrier (BBB) needs to be facilitated, for example through interaction with the transferrin receptor (TfR). Thus, a Fab fragment of the TfR antibody 8D3 was fused with two single chain variable fragments (scFv) of the Aβ protofibril selective antibody mAb158. Five Tribody proteins (A1-A5) were generated with different linkers between the Fab-8D3 and scFv-158. All proteins bound to TfR and Aβ protofibrils in vitro. Three of the proteins (A1-A3) were radiolabeled with iodine-125 and studied ex vivo in wild-type (wt) and transgenic mice overexpressing human Aβ. The systemic pharmacokinetics were similar with half-lives in blood of around 9h for all three ligands. Brain concentrations at 2h were around 1% of the injected dose per gram brain tissue, which is similar to what is observed for small molecular radioligands and at least 10-fold higher than antibodies in general. At 72h, transgenic mice showed higher concentrations of radioactivity in the brain than wt mice (12, 15- and 16-fold for A1, A2 and A3 respectively), except in the cerebellum, an area largely devoid of Aβ pathology. A3 was then labelled with iodine-124 for in vivo positron emission tomography (PET) imaging. Brain concentrations were quantified in six different regions showing a clear distinction both quantitatively and visually between wt and transgenic mice and a good correlation with Aβ pathology. We have thus produced a recombinant, bispecific protein, actively transported into the brain, for PET imaging within the CNS. In a longer perspective, this technique may enable imaging of other proteins involved in neurodegenerative diseases for which imaging agents are completely lacking today.
抗体对其靶分子具有高度特异性，但由于其脑穿透性差，限制了它们作为正电子发射断层扫描（PET）配体用于成像中枢神经系统（CNS）内的靶标。本研究旨在开发一种基于抗体的放射性配体，使用 Tribody 格式，用于正电子发射断层扫描（PET）成像可溶性淀粉样β（Aβ）原纤维，这些原纤维被认为是阿尔茨海默病中神经退行性变的原因。抗体，即使以较小的工程化形式表达，也是大分子，其不能以足够的量进入大脑用于成像目的。因此，需要促进其穿过血脑屏障（BBB）的转运，例如通过与转铁蛋白受体（TfR）相互作用。因此，TfR 抗体 8D3 的 Fab 片段与 Aβ原纤维选择性抗体 mAb158 的两个单链可变片段（scFv）融合。用不同的接头生成了 5 种 Tribody 蛋白（A1-A5）Fab-8D3 和 scFv-158 之间。所有蛋白均在体外与 TfR 和 Aβ原纤维结合。用碘-125 标记了其中的 3 种蛋白（A1-A3），并在野生型（wt）和过表达人 Aβ的转基因小鼠中进行了离体研究。三种配体的系统药代动力学相似，血液半衰期约为 9 小时。在 2 小时时，大脑浓度约为每克脑组织注射剂量的 1%，这与小分子放射性配体相似，至少比一般抗体高 10 倍。在 72 小时时，与野生型小鼠相比，转基因小鼠的大脑中放射性活性浓度更高（A1、A2 和 A3 分别为 12、15 和 16 倍），小脑除外，小脑是 Aβ病理区的大部分区域。然后用碘-124 标记 A3 进行体内正电子发射断层扫描（PET）成像。在六个不同区域定量检测脑浓度，在野生型和转基因小鼠之间在数量和视觉上均有明显区分，与 Aβ病理有很好的相关性。因此，我们已经生产了一种重组的双特异性蛋白，可主动转运到大脑中，用于 CNS 内的 PET 成像。从长远来看，这项技术可以实现对其他涉及神经退行性疾病的蛋白质的成像，而目前这些疾病完全缺乏成像剂。
