An Affibody Molecule Is Actively Transported into the Cerebrospinal Fluid via Binding to the Transferrin Receptor
Abstract
:1. Introduction
2. Results
2.1. Design and Production of scFv8D3-ZSYM73-ABD and ZSYM73-ABD
2.2. SPR Assays for Analysis of the Interaction between Recombinant Mouse TfR, MSA, and Aß1-40 with the Trispecific Fusion Protein
2.3. Flow Cytometry for Analysis of the Interaction between the Trispecific Fusion Protein and Mouse TfR-Expressing Endothelial Cells
2.4. Bioavailability of the Affibody Fusion Proteins in Mouse CSF
3. Discussion
4. Materials and Methods
4.1. Design and Molecular Cloning of Affibody Fusion Proteins
4.2. Protein Expression, Purification, and Quality Control
4.3. Analysis of Binding to Recombinant Proteins Using SPR-Based Biosensor Assays
4.4. Analysis of Binding to TfR on Mouse Brain Endothelial Cells Using Flow Cytometry
4.5. Analysis of CNS Uptake in a Mouse Model
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Immobilization Level | Rmax (RU) | ka (1/Ms) | kd (1/s) | KD (nM) |
---|---|---|---|---|
Surface 1: High (747 RU) | 73.3 ± 9.8 | 1.4 ± 0.1 × 104 | 3.5 ± 0.1 × 10−5 | 2.3 ± 0.02 |
Surface 2: Intermediate (487 RU) | 61.3 ± 19.4 | 1.2 ± 0.3 × 104 | 7.6 ± 0.9 × 10−5 | 6.6 ± 1.2 |
Surface 3: Low (229 RU) | 21.9 ± 4.2 | 2.0 ± 0.3 × 104 | 9.9 ± 0.5 × 10−5 | 5.2 ± 0.6 |
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Meister, S.W.; Hjelm, L.C.; Dannemeyer, M.; Tegel, H.; Lindberg, H.; Ståhl, S.; Löfblom, J. An Affibody Molecule Is Actively Transported into the Cerebrospinal Fluid via Binding to the Transferrin Receptor. Int. J. Mol. Sci. 2020, 21, 2999. https://doi.org/10.3390/ijms21082999
Meister SW, Hjelm LC, Dannemeyer M, Tegel H, Lindberg H, Ståhl S, Löfblom J. An Affibody Molecule Is Actively Transported into the Cerebrospinal Fluid via Binding to the Transferrin Receptor. International Journal of Molecular Sciences. 2020; 21(8):2999. https://doi.org/10.3390/ijms21082999
Chicago/Turabian StyleMeister, Sebastian W., Linnea C. Hjelm, Melanie Dannemeyer, Hanna Tegel, Hanna Lindberg, Stefan Ståhl, and John Löfblom. 2020. "An Affibody Molecule Is Actively Transported into the Cerebrospinal Fluid via Binding to the Transferrin Receptor" International Journal of Molecular Sciences 21, no. 8: 2999. https://doi.org/10.3390/ijms21082999