Identification of HLA-Cw6.02 and HLA-Cw7.01 allele-specific binding motifs by screening synthetic peptide libraries

Abstract

Unlike HLA-A and HLA-B, few peptide epitope motifs have been reported for HLA-C molecules. However, a number of cytotoxic T-lymphocyte epitopes derived from tumor antigens that bind to HLA-C molecules have been described. Here we report peptide-binding motifs for both HLA-Cw6.02 and HLA-Cw7.01 molecules. Recombinant human HLA molecules were generated and used to screen combinatorial 9mer peptide libraries. Complexes of HLA molecules properly folded and associated with beta2-microglobulin and peptides were identified using a conformation-specific HLA class I antibody conjugated to alkaline phosphatase. In the presence of substrate, peptide beads can be readily isolated and microsequenced to determine peptide identity. Of the peptides that bound to HLA-Cw6.02 and HLA-Cw7.01, 19 and 18 peptides, respectively, were sequenced, allowing motif identification for each C allele. This is the first report of an HLA-Cw7.01 peptide motif and extends the findings of Falk et al. [(1993) Proc Natl Acad Sci USA 90:12005] for an HLA-Cw6.02 motif. Anchoring amino acids for the HLA-Cw6.02 motif were phenylalanine or tyrosine in position (P)1, arginine in P2, and an aliphatic/aromatic residue at P9. Anchoring residues for HLA-Cw7.01 were positively charged amino acids in P1 and P2. Unlike most other HLA molecules, we were unable to assign P9 an anchoring residue, and we suspect that HLA-Cw7.01 binds peptides in an unconventional manner. Additionally, preferred amino acids were identified for both molecules. Identification of HLA-Cw6.02 and HLA-Cw7.01 peptide-binding motifs makes a significant contribution to the C allele peptide-binding motifs and will allow investigators to predict, design, and test HLA-Cw6.02 and HLA-Cw7.01 engineered peptides for immunotherapy.

Peptide-specific engagement of the activating NK cell receptor KIR2DS1

“…The peptides included 14 of the 19 previously described synthetic HLA-C*06:02-binding peptides and 6 novel HIV-1-derived peptides. Most of the peptides stabilizing HLA-C*06:02 showed the same binding motif consisting of a phenylalanine in position 1 of the peptide sequence (15/20), an arginine in position 2 of the peptide sequence (15/20) and an aliphatic amino acid (valine, leucine or isoleucine) in position 9 of the peptide sequence (16/20), which is in agreement with the previously defined binding motif for HLA-C*06:02 26–28 (see Supplementary Table 1). …”

“…We first tested 19 synthetic peptides that had been previously described to bind to HLA-C*06:02 26 . Furthermore, 568 overlapping peptides spanning the entire HIV-1 clade B sequence peptides (346 18aa-long peptides covering the entire HIV-1 consensus sequence and 222 decametric peptides overlapping by 9 amino acid and covering p24 GAG) were assessed for their potential to stabilize HLA-C*06:02.…”

Uncovering the Peptide-Binding Specificities of HLA-C: A General Strategy To Determine the Specificity of Any MHC Class I Molecule

“…The motifs identified in the present study largely match those previously described for HLA-C*03:04 (9), HLA-C*06:02 (39,40), HLA-C*07:01 (40), and HLA-C*07:02 (39), supporting the notion that the approach taken in the present study yields an accurate representation of the peptide-binding properties of HLA-I molecules. For all HLA-C allotypes, whether tested experimentally or inferred bioinformatically, we observed the presence of a strong and dominant C-terminal primary anchor position, which encompassed a rather restricted repertoire of hydrophobic and aromatic amino acid residues (Phe, Ile, Leu, Met, Val, and Tyr).…”

“…HLA-C*02:02, -C*03:03, -C*03:04, -C*04:01, -C*05:01, -C*06:02, -C*07:02, -C*07:04, -C*08:01, and -C*12:03 have an auxiliary anchor at P1 favoring Tyr or Phe/Tyr, whereas HLA-C*07:01 and -C*15:02 have an auxiliary anchor at P1 favoring the positively charged Lys and Arg residues. Whereas the P1 and P2 pockets of the latter group have a net negative charge, which confers a preference for positive amino acids at P1 and P2 (40), the former group has an Asn 66 reducing the negative charge of their P1 and P2 pockets (Supplemental Table IV), which is likely to shift their preference from positive toward noncharged amino acids. As alluded to above, HLA-C*05:01 and -C*08:02 show auxiliary preference for Ala and Ser in position 2.…”

HLA-A*01:03, HLA-A*24:02, HLA-B*08:01, HLA-B*27:05, HLA-B*35:01, HLA-B*44:02, and HLA-C*07:01 Monochain Transgenic/H-2 Class I Null Mice: Novel Versatile Preclinical Models of Human T Cell Responses

“…The lack of response to the HIV nef 186–194 peptide likely reflects the fact that this peptide is deprived of the HLA-A*24:02 binding motifs (P2, Y/F, P9, F/W/I/L) and, as such, is predicted as a nonbinder peptide by all epitope prediction programs. A similar argument certainly applies to the three published HLA-C*07:01 epitopes that all lack the positively charged residues in P1 and P2 considered as anchoring residues for HLA-C*07:01, but this argument remains somewhat hypothetical because the HLA-C*07:01 peptide-binding motifs have only been partially identified (63). …”