These peptides share the common motif ‘IMYNYPAM’ Quizartinib and bound to six out of eight alleles (i.e. HLA-A*0201, A*0301, A*1101, A*2401, B*0702 and B*1501). At the C-terminus, we identified a different motif, MMARDTAE, shared by the peptides AMMARDTAE (TB10.482–90) and MMARDTAEA (TB10.483–91). This motif bound to three out of eight alleles (HLA-A*0201, B*0702 and B*1501; Table 1). We chose TB10.4 peptides and performed affinity (ED50) and off-rate (t1/2) analysis for (i) peptides identified as binders (above 20% compared with the positive control peptide), and (ii) MHC class I-binding epitopes below the 20% cut-off if they represented

the only peptides that bound to MHC class I alleles; for example, AMMARDTAE and MMARDTAEA for A*0101, and MMARDTAEA for B*0801. Affinity between candidate peptides and the respective MHC class I complex was found to be in the range of 60 nm to 800 μm, with the majority (75%) in the range of 1–80 μm. Different TB10.4 peptides bound with different affinity

to the same MHC allele; for example, the peptide QIMYNYPAM (TB10.43–11) bound with an affinity of 800 μm to the allele HLA-B*0702, while the peptide AMMARDTAE (TB10.482–90) bound with an affinity of 80 nm to the same MHC class I allele. Also, the identical peptide could bind with different affinity to different MHC class I alleles. For example, the peptide IMYNYPAML I-BET-762 clinical trial (TB10.44–12) bound to HLA-A*0201 with an affinity of 800 nm, to A*0301 with an affinity of 700 nm, to A*2402 with an

affinity of 100 nm, to B*0702 with an affinity of 30 μm and to B*1501 with an affinity of 20 μm. Overall, the TB10.4 peptides bound with higher affinity to HLA-A alleles than to HLA-B alleles (Fig. 3, Table 2). The off-rate assay was used to evaluate the relative stability of each MHC class I complex. The dissociation rate of the peptides spanned a wide range of < 1 to 27 hr, with the majority of epitopes (27 of 52) in the range of 1–3 hr. Four peptides, for example HLA-B*0702 RAYHAMSST (TB10.467–75), exhibited a dissociation rate of < 1 hr, while nine of 52 peptides showed a t1/2 value of more than 5 hr, for example HLA-A*0201 AMMARDTAE (TB10.482–90). We could identify differences both (i) within a single MHC class I allele presenting different Ureohydrolase peptides, for example HLA-A*0201 which presents the peptide IMYNYPAML (TB10.44–12) with an off-rate of approximately 27 hr and GITYQAWQA (TB10.448–56) with an off-rate of 0·7 hr, and (ii) between different alleles presenting identical peptides, for example the peptide IMYNYPAML (TB10.44–12) which exhibited an off-rate of approximately 27 hr for HLA-A*0201, approximately 1 hr for A*0301, approximately 1·5 hr for A*2402/B*0702 and approximately 4 hr for B*1501. We could not find any correlation between affinity and off-rate; some peptides with high affinity had very long off-rates, while other peptides showed the opposite dissociation pattern (Fig. 3 and Table 2).