“Endothelial Antibody Factory” at the Blood Brain Barrier: Novel Approach to Therapy of Neurodegenerative Diseases
Abstract
:1. Introduction
2. Materials and Methods
2.1. Synthesis of Tetrapalmitoyl-Tris-Lysine-β-Amyloid-Peptide and TDP-43-Peptide
2.2. Preparation of the Antigens
2.3. Immunizations, Antibody Fragments (Fabs) Cloning and Production
2.4. Solubilization of Anti-β-Amyloid and Anti-TDP-43 Aggregates by Antibodies and Antibody Fragments
2.5. Plasmids
2.6. Cell Line
2.7. Cell Transduction with tdTomato Expression Lentiviral Vectors
2.8. Cell Transfection and Cloning
2.9. Western Blot His-Tagged Fab
2.10. Assessment of Surface Markers Using Flow Cytometry
2.11. Hypoxia Sensitivity of Murine Brain-Derived Endothelial Cells (MBrMECs) Recognition by MAgEC 10.5 RT Cell Line by Adhesion Experiment
2.12. Endothelial Progenitor Cells Recruitment Assays by BBB Brain Spheroid
2.13. Animal Experiments and Immunofluorescence
2.14. Statistics
3. Results
3.1. New Antibodies and Antibody Fragments Directed against β-Amyloid and TDP-43 Proteins
3.2. Construction of Expression Vectors Encoding Anti-β-Amyloid and TDP-43 Fabs
3.3. Preliminary Study with Stable Expression of s-EGFP and Transient Expression of Fab
3.4. Creation and Characterization of the MAgEC 10.5 RT Anti-β-Amyloid and MAgEC 10.5 RT Anti-TDP-43 Cells: Solubilizing Fab Production and Cell Markers
3.5. Endothelial Progenitor Cells Recruitment Assays by BBB Brain Spheroid
3.6. Homing, Adhesion and Integration of the Injected Cells into the Brain Vasculature
3.7. Exogenic TDP-43 Antibody Secreted by Therapeutic MAgEC Cells Observed in the Brain Vasculature and Brain Parenchyma
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Primary Antibodies | Catalog Number |
---|---|
Anti-Histag Chimeric | Merck, SAB5600096 |
Anti-Mouse IgG (Fab specific) | ThermoFisher, 31413 |
Secondary antibodies | |
Anti-Histag Chimeric secondary | Merck, A1293 |
Rabbit anti-Goat IgG (H + L) secondary | ThermoFisher, 81-1620 |
Antibody | Catalog Number | Details |
---|---|---|
CD34 | 152207 (Biolegend) | Rat IgG2a Brilliant Violet 421™ |
CD45 | 103111(Biolegend) | Rat IgG2b APC |
UEA-1 | DL-1069 (Vector Labratories) | DyLight649 labeling |
Isotype Control Antibody | 400511 (Biolegend) | Rat IgG2a APC |
Isotype Control Antibody | 400611(Biolegend) | Rat IgG2b APC |
Clone Name | Primary Heavy Chain Protein Sequence | |||
---|---|---|---|---|
HC Type | Sig. Pep. | VH | Constant | |
anti-TDP-43 antibody (cAb2508) | Mouse IgG2b | MEWIWIFLFILSGTAGVQS | QVQLQQSGAELARPGASVKLSCKASGYTFTSYGISWVRQRTGQGLEWIGEIYPRRGNTYYNEKFKGKATLTAYKSSGTAYMELRSLTSEDSAVFFCARGGIYYGNLFDYWGQGTTLTVSS | AKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVRISWFVNNVEVHTAQTQTHREDYNSTIRVVSALPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLDIKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK |
Primary light chain protein sequence | ||||
LC type | Sig. pep. | VL | Constant | |
Mouse lambda | MAWISLILSLLALSSGAIS | QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWFSNHWVFGGGTKLTVLG | QPKSSPSVTLFPPSSEELETNKATLVCTITDFYPGVVTVDWKVDGTPVTQGMETTQPSKQSNNKYMASSYLTLTARAWERHSSYSCQVTHEGHTVEKSLSRADCS | |
Primary heavy chain protein sequence | ||||
HC type | Sig. pep. | VH | Constant | |
anti-β-Amyloid antibody (cAb2789) | Mouse IgM | MEWPLIFLFLLSGTAGVQS | QVQLQQSGAELVKPGASVKISCKASGYAFSNYWMNWVKQRPGKGLEWIGQIYPGDGDTNYNGKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYFCARGDYWGQGTTLTVSS | ESQSFPNVFPLVSCESPLSDKNLVAMGCLARDFLPSTISFTWNYQNNTEVIQGIRTFPTLRTGGKYLATSQVLLSPKSILEGSDEYLVCKIHYGGKNKDLHVPIPAVAEMNPNVNVFVPPRDGFSGPAPRKSKLICEATNFTPKPITVSWLKDGKLVESGFTTDPVTIENKGSTPQTYKVISTLTISEIDWLNLNVYTCRVDHRGLTFLKNVSSTCAASPSTDILTFTIPPSFADIFLSKSANLTCLVSNLATYETLNISWASQSGEPLETKIKIMESHPNGTFSAKGVASVCVEDWNNRKEFVCTVTHRDLPSPQKKFISKPNEVHKHPPAVYLLPPAREQLNLRESATVTCLVKGFSPADISVQWLQRGQLLPQEKYVTSAPMPEPGAPGFYFTHSILTVTEEEWNSGETYTCVVGHEALPHLVTERTVDKSTGKPTLYNVSLIMSDTGGTCY |
Primary light chain protein sequence | ||||
LC type | Sig. pep. | VL | Constant | |
Mouse kappa | MESQTQVLMFLLLWVSGACA | DIVMTQSPSSLAMSVGQKVTMSCKSSQSLLNSSNQKNYLAWYQQKPGQSPKLLVYFASTRESGVPDRFIGSGSGTDFTLTISSVQAEDLADYFCQQHYNTPLTFGAGTKLELK | RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC |
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Thinard, R.; Farkas, A.E.; Halasa, M.; Chevalier, M.; Brodaczewska, K.; Majewska, A.; Zdanowski, R.; Paprocka, M.; Rossowska, J.; Duc, L.T.; et al. “Endothelial Antibody Factory” at the Blood Brain Barrier: Novel Approach to Therapy of Neurodegenerative Diseases. Pharmaceutics 2022, 14, 1418. https://doi.org/10.3390/pharmaceutics14071418
Thinard R, Farkas AE, Halasa M, Chevalier M, Brodaczewska K, Majewska A, Zdanowski R, Paprocka M, Rossowska J, Duc LT, et al. “Endothelial Antibody Factory” at the Blood Brain Barrier: Novel Approach to Therapy of Neurodegenerative Diseases. Pharmaceutics. 2022; 14(7):1418. https://doi.org/10.3390/pharmaceutics14071418
Chicago/Turabian StyleThinard, Reynald, Attila E. Farkas, Marta Halasa, Melanie Chevalier, Klaudia Brodaczewska, Aleksandra Majewska, Robert Zdanowski, Maria Paprocka, Joanna Rossowska, Lam Tri Duc, and et al. 2022. "“Endothelial Antibody Factory” at the Blood Brain Barrier: Novel Approach to Therapy of Neurodegenerative Diseases" Pharmaceutics 14, no. 7: 1418. https://doi.org/10.3390/pharmaceutics14071418