Selection of single domain anti-transferrin receptor antibodies for blood-brain barrier transcytosis using a neurotensin based assay and histological assessment of target engagement in a mouse model of Alzheimer's related amyloid-beta pathology.
Selection of single domain anti-transferrin receptor antibodies for blood-brain barrier transcytosis using a neurotensin based assay and histological assessment of target engagement in a mouse model of Alzheimer's related amyloid-beta pathology.
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DOI：
10.1371/journal.pone.0276107
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发表时间：
2022
期刊：
影响因子：
3.7
通讯作者：
中科院分区：
文献类型：
--
作者：
血脑屏障（BBB）是开发用于许多神经系统疾病的特异性诊断成像剂的主要障碍。在这项研究中，我们的目的是产生单域抗小鼠转铁蛋白受体抗体（抗mTfR VHH）介导血脑屏障转胞吞作用的新的MRI分子造影剂的组成部分。通过用mTfR免疫美洲驼、产生VHH噬菌体展示文库、免疫淘选和候选物的体外表征来产生抗mTfR VHH。定点诱变用于产生另外的变体。VHH与神经降压素（NT）的融合物允许在野生型小鼠中快速地、基于低血糖的VHH介导的BBB转胞吞作用的筛选。将一种抗mTfR VHH变体与抗淀粉样蛋白β（Aβ）VHH二聚体融合，并用荧光染料标记，以直接评估AD相关Aβ斑块病理学小鼠模型中的体内靶点结合。称为M1和变体的抗mTfR VHH与mTfR的结合亲和力为<1 nM至1.52 nM。VHH与mTfR结合的亲和力与静脉内注射600 nmol/kg体重后VHH-NT诱导的低温效应的效率相关，范围从非结合突变体的不可检测到最佳突变体的-6 ° C。将具有最强低温效应的抗mTfR VHH变体M1 P96 H与抗A β VHH二聚体融合并用Alexa 647标记;染料标记的VHH融合构建体在低至0.22 nM的浓度下仍结合mTfR和Aβ斑块。然而，在以600 nmol/kg体重静脉注射到APP/PS1转基因小鼠中后，没有检测到高于对照水平的斑块标记。因此，NT诱导的低温与皮质中的直接靶点接合不相关，可能是因为NT诱导的低温所需的浓度低于产生原位标记所需的浓度。这些发现揭示了NT诱导的低温（可能由下丘脑介导）与皮质中Aβ斑块的直接参与之间的重要分离。评估抗mTfR VHH BBB转胞吞作用的其他方法将需要开发用于抗mTfR VHH筛选和新型MRI分子造影剂的开发。
The blood-brain barrier (BBB) presents a major obstacle in developing specific diagnostic imaging agents for many neurological disorders. In this study we aimed to generate single domain anti-mouse transferrin receptor antibodies (anti-mTfR VHHs) to mediate BBB transcytosis as components of novel MRI molecular contrast imaging agents. Anti-mTfR VHHs were produced by immunizing a llama with mTfR, generation of a VHH phage display library, immunopanning, and in vitro characterization of candidates. Site directed mutagenesis was used to generate additional variants. VHH fusions with neurotensin (NT) allowed rapid, hypothermia-based screening for VHH-mediated BBB transcytosis in wild-type mice. One anti-mTfR VHH variant was fused with an anti-amyloid-beta (Aβ) VHH dimer and labeled with fluorescent dye for direct assessment of in vivo target engagement in a mouse model of AD-related Aβ plaque pathology. An anti-mTfR VHH called M1 and variants had binding affinities to mTfR of <1nM to 1.52nM. The affinity of the VHH binding to mTfR correlated with the efficiency of the VHH-NT induced hypothermia effects after intravenous injection of 600 nmol/kg body weight, ranging from undetectable for nonbinding mutants to -6°C for the best mutants. The anti-mTfR VHH variant M1P96H with the strongest hypothermia effect was fused to the anti-Aβ VHH dimer and labeled with Alexa647; the dye-labeled VHH fusion construct still bound both mTfR and Aβ plaques at concentrations as low as 0.22 nM. However, after intravenous injection at 600 nmol/kg body weight into APP/PS1 transgenic mice, there was no detectible labeling of plaques above control levels. Thus, NT-induced hypothermia did not correlate with direct target engagement in cortex, likely because the concentration required for NT-induced hypothermia was lower than the concentration required to produce in situ labeling. These findings reveal an important dissociation between NT-induced hypothermia, presumably mediated by hypothalamus, and direct engagement with Aβ-plaques in cortex. Additional methods to assess anti-mTfR VHH BBB transcytosis will need to be developed for anti-mTfR VHH screening and the development of novel MRI molecular contrast agents.
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影响因子：
3.8
作者：
Boado, Ruben J.;Zhang, Yun;Wang, Yuntao;Pardridge, William M.
通讯作者：
Pardridge, William M.
影响因子：
3.2
作者：
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通讯作者：
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影响因子：
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作者：
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通讯作者：
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影响因子：
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作者：
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通讯作者：
Stanimirovic, Danica B.
影响因子：
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作者：
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通讯作者：
Brody DL