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• W4377029015 abstract "<b>Abstract ID 18127</b> <b>Poster Board 125</b> The Blood-Brain Barrier (BBB) protects the human brain by restricting the access of foreign substances to the brain, including even beneficial therapeutic agents with extremely low permeability. Receptor-mediated transcytosis (RMT) is a pathway to transport macromolecules such as antibodies and peptides across the BBB., which has been widely studied as a drug delivery strategy to overcome the BBB. We utilized a novel T-cell receptor mimic antibody (TCRm) to target brain endothelial cells with high affinity and specificity. The TCRm RL6A has been generated to recognize a peptide epitope derived from p68 RNA helicase (YLLPAIVHI) presented by MHC class-I subtype, HLA-A2, which is highly expressed by brain endothelial cells. Our lab previously showed the binding and internalization of RL6A in both <i>in-vitro</i> and <i>in vivo</i> brain endothelial cells. Therefore, RL6A could be a potential vector for brain drug delivery. Based on the preliminary findings, we hypothesized that the upregulated cellular peptide processing and MHC-I expression levels by Interferon gamma (IFN-γ) will increase the internalization of RL6A in brain endothelial cells. Additionally, we have speculated that RL6A antibodies enter the brain endothelial cells via Clathrin-Independent Endocytosis (CIE), a characteristic of MHC I endocytosis. We studied these mechanisms on hCMEC/D3 cells, an immortalized human brain endothelial cell line, and on human induced pluripotent stem cell (iPSC) derived brain microvascular endothelial cells (hBMEC). Cells were pre-incubated with antibodies at 4°C for 30min and the internalization was initiated by adding warmed media, and then cells were incubated at 37°C at different time points up to 120 min. Then, cells were further processed for analysis by flow cytometry or confocal miscroscopy. Our flow cytometry data showed that RL6A internalized from the surface of hCMEC/D3 cells in a biphasic pattern with an initial rapid phase (>30% internalized within 10 min) followed by a slower phase at later time points. Furthermore, the mean fluorescence intensity (MFI) for surface-bound RL6A increased approximately two-fold for the interferon-gamma (IFN-γ) treated cells in both hCMEC/D3 and hBMEC cells. For the trafficking study in hCMEC/D3 cells by confocal microscopy, we used pixel-based and object-based analysis in AIVIA software to quantify the colocalization fraction between RL6A and endogenous early endosome proteins. The results showed that the fraction of RL6A colocalized with the small GTPase ARF6 increased from 6.5% ± 9.6% (mean ± SD) at 10 min to a maximum of 34.3% ±14.2% at 30min, and then gradually decreased at 60 min and 120 min (11.1% ± 7.8%, p<0.0001 vs 30 min). The time course for RL6A colocalization with Rab5 was similar to ARF6, but the maximum reached only 11.3% at 30 min. The colocalization of RL6A and early endosomal marker EEA1 showed slower dynamics, with a peak fraction of 5.7% ± 3.5% at 60 min. The absolute number of internalized RL6A reached a plateau after 60 min. In conclusion, TCRm RL6A internalization occurred mainly in the first 30 min after endocytosis initiation. IFN-γ treatment upregulated the expression level of the YLL-A2 complex and increased the RL6A binding and internalization for both hCMEC/D3 and iPSC-derived hBMEC cells. The colocalization analysis expanded on our previous results and demonstrated that RL6A undergoes CIE." @default.
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• W4377029015 date "2023-05-18" @default.
• W4377029015 modified "2023-09-29" @default.
• W4377029015 title "Trafficking pathway of T-cell receptor mimic antibodies (TCRm) RL6A in human brain-derived endothelial cells" @default.
• W4377029015 doi "https://doi.org/10.1124/jpet.122.181270" @default.
• W4377029015 hasPublicationYear "2023" @default.
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