Real time detection of peptide-MHC dissociation reveals that improvement of primary MHC-binding residues can have a minimal, or no, effect on stability

Abstract

The majority of known major histocompatibility complex class I (MHCI)-associated tumor-derived peptide antigens do not contain an optimal motif for MHCI binding. As a result, anchor residue-modified 'heteroclitic' peptides have been widely used in therapeutic cancer vaccination trials in order to enhance immune responsiveness. In general, the improved stability of these heteroclitic complexes has been inferred from their improved immunogenicity but has not been formally assessed. Here, we investigated the binding of 4 HLA A*0201-restricted tumor-derived peptides and their commonly used heteroclitic variants. We utilized a cell surface binding assay and a novel robust method for testing the durability of soluble recombinant pMHCI in real time by surface plasmon resonance. Surprisingly, we show that heteroclitic peptides designed with optimal MHC binding motifs do not always form pMHCs that are substantially more stable than their wildtype progenitors. These findings, combined with our recent discovery that TCRs can distinguish between wildtype peptides and those altered at a primary buried MHC anchor residue, suggest that altered TCR binding may account for a large part of the increased immune response that can be generated by anchor residue-modified ligands. Our results further highlight the fact that heteroclitic peptide-based immune interventions require careful evaluation to ensure that wildtype antigen specificity is maintained in vivo.

Fig. 1

FACS analysis of pMHCI binding using TAP deficient T2 cells. We used TAP deficient T2 cells, which lack the ability to transport peptide fragments to the endoplasmic reticulum to form stable pMHCI, to analyze the stability of the natural and heteroclitic pMHCIs using FACS analysis. No peptide, and the HLA-B*3501-restricted EBNA-1_407–415 (HPVGEADYF) peptide were used as negative controls of binding, and the HLA A*0201-restricted influenza M1_58–66 (GILGFVFTL) peptide was used as a positive control of binding. The mean fluorescence intensity (MFI) of cell surface pMHCI staining with an anti-human HLA A2:RPE antibody revealed that the natural tumor antigen, KIFGSLAFL, was more stable than the heteroclitic variant, KLFGSLAFV, whereas the natural tumor peptides SLLMWITQC, YLEPGPVA and VISNDVCAQV were less stable than their heteroclitic counterparts: SLLMWITQL, YLEPGPVV and VLSNDVCAQV. Data at a peptide concentration of 100 μM are shown. The binding assay was performed at a range of peptide concentrations (100 μM, 50 μM, 10 μM and 1 μM). LogEC50s (M) for this range are shown in Table 1. The mean standard deviation, representing 4 separate experiments, is shown as error bars.

Fig. 2

Real time SPR pMHC stability assay. We immobilized each wildtype and heteroclitic pMHCI pair to the surface of a CM5 chip using streptavidin-biotin coupling to assess the half-life of each complex in real time. In a similar trend to the T2-binding assay, we observed that; (A) the natural tumor antigen, HLA A*0201-KIFGSLAFL, had a longer half-life compared to the heteroclitic variant, (A) HLA A*0201-KLFGSLAFV, whereas the natural tumor antigens (B) HLA A*0201-SLLMWITQC, (C) HLA A*0201-VISNDVCAQV and (D) HLA A*0201-YLEPGPVA had faster off-rates compared to their heteroclitic counterparts, (B) HLA A*0201-SLLMWITQL, (C) HLA A*0201-VLSNDVCAQV and (D) HLA A*0201-YLEPGPVV. Data are representative of 3 separate experiments.