Fluorescence labeling of anchor-modified Mart-1 peptide for increasing its affinity for HLA-A*0201: Hit two targets with one arrow |
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Authors: | Pooya Fattahi Najmeh Salehi Zahra Azizi Javad Mohammadi Amir Norouzy Seyed Mohammad Moazzeni |
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Institution: | 1. Department of Immunology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran;2. Institute of Medical Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran;3. Department of Molecular Medicine, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran;4. Department of Biomedical Engineering, Faculty of New Sciences and Technologies, University of Tehran, Tehran, Iran;5. Bioprocess Engineering Department, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran |
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Abstract: | One of the most successful strategies in designing peptide-based cancer vaccines is modifying natural epitope peptides to increase their binding strength to human leukocyte antigens (HLAs). Anchor-modified Mart-1 peptide (ELAGIGILTV) is among the artificial epitope peptides with the highest binding affinity for HLA-A*0201. In this study, by fluorescence labeling of its either C- or N-terminus with Nε-(5-carboxyfluorescein)-l -lysine, we not only made it traceable but also drastically increased its binding strength to HLA-A*0201. HLA streptamer, for the first time, is introduced for measuring the binding constants (Ka) of the labeled peptides. The affinity of the labeled peptides for the HLA-A*201 of the MCF-7 cells was extraordinarily high and co-incubating them with the highest possible amount of the unlabeled peptide, as a competitor, did not significantly prohibit them from binding to the HLA. The reproducibility of the obtained results was confirmed by using the T2 cell line. The HLA-deficient K562 cell line was used as the negative control. With in silico simulations, we found two hydrophobic pockets on both sides of HLA-A*0201 for anchoring the C- or N-terminal 5-carboxyfluorescein probe, which can explain the extraordinary affinity of the labeled peptides for the HLA-A*0201. |
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Keywords: | cancer vaccine epitope peptide flow cytometry MCF-7 molecular dynamics supramolecular interaction |
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