Protein Drug Design Group, Department of Pharmacy, Uppsala University, Uppsala, Sweden.
Department of Public Health and Caring Sciences, Uppsala University, Uppsala, Sweden.
Sci Rep. 2022 Dec 12;12(1):21479. doi: 10.1038/s41598-022-25965-x.
The blood-brain barrier (BBB) greatly limits the delivery of protein-based drugs into the brain and is a major obstacle for the treatment of brain disorders. Targeting the transferrin receptor (TfR) is a strategy for transporting protein-based drugs into the brain, which can be utilized by using TfR-binding BBB transporters, such as the TfR-binding antibody 8D3. In this current study, we investigated if binding to heparan sulfate (HS) contributes to the brain uptake of a single chain fragment variable of 8D3 (scFv8D3). We designed and produced a scFv8D3 mutant, engineered with additional HS binding sites, HS(+)scFv8D3, to assess whether increased HS binding would improve brain uptake. Additionally, a mutant with a reduced number of HS binding sites, HS(-)scFv8D3, was also engineered to see if reducing the HS binding sites could also affect brain uptake. Heparin column chromatography showed that only the HS(+)scFv8D3 mutant bound HS in the experimental conditions. Ex vivo results showed that the brain uptake was unaffected by the introduction or removal of HS binding sites, which indicates that scFv8D3 is not dependent on the HS binding sites for brain uptake. Conversely, introducing HS binding sites to scFv8D3 decreased its renal excretion while removing them had the opposite effect.
血脑屏障（BBB）极大地限制了蛋白质类药物进入大脑，是治疗脑部疾病的主要障碍。靶向转铁蛋白受体（TfR）是将蛋白质类药物输送到大脑的一种策略，可以利用 TfR 结合 BBB 转运体，如 TfR 结合抗体 8D3。在本研究中，我们研究了与肝素硫酸酯（HS）结合是否有助于单链片段变量 8D3（scFv8D3）进入大脑。我们设计并生产了 scFv8D3 突变体，工程化了额外的 HS 结合位点，HS(+)scFv8D3，以评估增加 HS 结合是否会提高脑摄取。此外，还设计了一个 HS 结合位点减少的突变体，HS(-)scFv8D3，以观察减少 HS 结合位点是否也会影响脑摄取。肝素柱层析表明，只有 HS(+)scFv8D3 突变体能在实验条件下结合 HS。离体结果表明，引入或去除 HS 结合位点对脑摄取没有影响，这表明 scFv8D3 不依赖 HS 结合位点进入大脑。相反，向 scFv8D3 中引入 HS 结合位点会降低其肾脏排泄，而去除 HS 结合位点则会产生相反的效果。
