Fluorescence Labeling of Anchor-Modified Mart-1 Peptide for Increasing Its Affinity for Hla-A*0201: Hit Two Targets With One Arrow Publisher Pubmed



Fattahi P¹ ; Salehi N² ; Azizi Z³ ; Mohammadi J⁴ ; Norouzy A⁵ ; Moazzeni SM¹
Source: Journal of Peptide Science Published:2023

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. © 2023 European Peptide Society and John Wiley & Sons, Ltd.