To answer the question of whether affinity or stability is the better correlate of immunogenicity, we extracted 12 affinity-balanced pairs each consisting of an “immunogenic binder” and a “nonimmunogenic binder” according to Sette and colleagues [6]. These peptides were synthesized and affinity and stability of their interactions with HLA-A*02:01 was measured. This representative analysis showed that “immunogenic binders” were significantly more stably bound to HLA-A*02:01 than “nonimmunogenic binders” (p = 0.0007, paired two-tailed

Student’s t-test) (Table 1, Fig. 4B), whereas no significant difference in affinity was observed between the two groups (Table 1, Fig. 4A). Note that one of the reported immunogenic peptides, RTLLGLILFV, in our hands was a low-affinity, low-stability-binding peptide. Upon closer inspection, the N-terminally truncated peptide, TLLGLILFV, appeared to be a likely HLA-A*02:01-binding peptide. This peptide FG-4592 cost was synthesized and found to be a high-stability (half-life 33 h) peptide. We would like to suggest that TLLGLILFV is the real HLA-A*02:01-restricted CTL epitope. Depicting this data in a log(stability) versus log(affinity) plot showed that the increased

stability of peptide-HLA-A*02:01 complexes involving “immunogenic binders” (y = 0.65x − 5.1, R2 = 0.65) versus Doxorubicin research buy “nonimmunogenic binders” (y = 0.75x − 4.5, R2 = 0.53) was seen throughout the binding range KD < 100 nM (Fig. 4C). When we inspected the 2 × 12 affinity-paired peptides (24 ever in total), we noted that 10 of 12 peptides with optimal amino acids residues in both anchor position 2 (LM) and C-terminal (VLI) had a half-life of more than 5 h, whereas nine of 12 peptides with a suboptimal amino acid residue (typically T or Q in position 2 or C-terminally) had a half-life of less than 5 h. At face value, this highly significant distribution (p = 0.014, Chi-square test with Yates correction) suggests that peptide-HLA-A*02:01 complexes are destabilized by just

one of the anchor positions being occupied with a suboptimal amino acid. For the seven peptides with suboptimal anchor residues, we substituted the suboptimal anchor residue with an optimal residue (leucine or methionine in position 2 and valine in C-terminal), and repeated the stability experiment. In all seven cases, the stability was improved (in six of the seven peptides, stability was increased by seven to tenfold), and four of the seven previously unstable peptides achieved a half-life better than 5 h, see Table 2. Thus, there appear to be a subtle difference in the specificity of high-affinity peptides, which may tolerate a suboptimal amino acid residue in an anchor position, and the specificity of high-stability peptides, which seems to be less inclined to tolerate suboptimal amino acid residue in anchor positions (in particular not in position 2).