A novel vascular targeting strategy for brain-derived endothelial cells using a TCR mimic antibody.
Organ-specific vascular targeting, for example, to the blood-brain barrier, requires the identification of unique molecular addresses on a subset of endothelial cells. The present study describes a crucial step towards tapping the exquisite specificity of the peptide/HLA class I system for this goal. We utilized a novel T-cell receptor (TCR) mimic antibody of high affinity and specificity, which is restricted by HLA-A2 and has been generated to recognize a peptide epitope derived from p68 RNA helicase (YLLPAIVHI). The parent protein is highly expressed by brain endothelial cells. Flow cytometry and confocal imaging showed that the antibody binds to HLA-A2-positive human brain-derived endothelial cells, both immortalized hCMEC/D3 cells and primary cells. The TCR mimic antibody undergoes internalization into vesicles, where significant colocalization occurs with the early endosomal marker EEA-1, but barely with caveolin-1. To our knowledge internalization of neither MHC class I protein nor TCR mimics by brain endothelial cells has been previously observed. Knock down of p68 protein expression by siRNA reduced the presentation of YLLPAIVHI-peptide/HLA-A2 complexes on the cell membrane by half as measured by flow cytometry 48 h later. We also found that brain endothelial cells isolated from HLA-A2 transgenic mouse strains express the A2 transgene, and brain endothelial cells of one of these strains also present YLLPAIVHI-peptide/HLA-A2, making these mouse strains suitable models for studying TCR mimic antibodies in vivo. In conclusion, these data strongly support the notion that TCR mimic antibodies could be a new class of therapeutic targeting agents in a wide variety of diseases.
Bhattacharya
Raktima
R
Department of Pharmaceutical Sciences, Center for Vascular Drug Research, Texas Tech University Health Sciences Center, Amarillo, Texas 79106, USA.
Xu
Yan
Y
Rahman
Md Ashequr
MA
Couraud
Pierre-Olivier
PO
Romero
Ignacio A
IA
Weksler
Babette B
BB
Weidanz
Jon A
JA
Bickel
Ulrich
U
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D000915:Antibody Affinity; D000918:Antibody Specificity; D001812:Blood-Brain Barrier / Q000166:cytology / Q000276:immunology* / Q000378:metabolism; D001921:Brain / Q000098:blood supply*; D051242:Caveolin 1 / Q000378:metabolism; D002478:Cells, Cultured; D053487:DEAD-box RNA Helicases / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D004705:Endocytosis; D011992:Endosomes / Q000276:immunology / Q000378:metabolism; D042783:Endothelial Cells / Q000276:immunology* / Q000378:metabolism; D005434:Flow Cytometry; D015789:HLA-A2 Antigen / Q000235:genetics / Q000276:immunology*; D006801:Humans; D018189:Immunomagnetic Separation; D051379:Mice; D008810:Mice, Inbred C57BL; D016688:Mice, Inbred NOD; D008822:Mice, Transgenic; D018613:Microscopy, Confocal; D018716:Molecular Mimicry; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000276:immunology; D034622:RNA Interference; D011948:Receptors, Antigen, T-Cell / Q000276:immunology* / Q000378:metabolism; D033921:Vesicular Transport Proteins / Q000378:metabolism
D000911:Antibodies, Monoclonal; C495540:CAV1 protein, human; D051242:Caveolin 1; D015789:HLA-A2 Antigen; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D011948:Receptors, Antigen, T-Cell; D033921:Vesicular Transport Proteins; C093846:early endosome antigen 1; D053487:DEAD-box RNA Helicases