Limits...
Direct identification of clinically relevant neoepitopes presented on native human melanoma tissue by mass spectrometry

View Article: PubMed Central - PubMed

ABSTRACT

Although mutations may represent attractive targets for immunotherapy, direct identification of mutated peptide ligands isolated from human leucocyte antigens (HLA) on the surface of native tumour tissue has so far not been successful. Using advanced mass spectrometry (MS) analysis, we survey the melanoma-associated immunopeptidome to a depth of 95,500 patient-presented peptides. We thereby discover a large spectrum of attractive target antigen candidates including cancer testis antigens and phosphopeptides. Most importantly, we identify peptide ligands presented on native tumour tissue samples harbouring somatic mutations. Four of eleven mutated ligands prove to be immunogenic by neoantigen-specific T-cell responses. Moreover, tumour-reactive T cells with specificity for selected neoantigens identified by MS are detected in the patient's tumour and peripheral blood. We conclude that direct identification of mutated peptide ligands from primary tumour material by MS is possible and yields true neoepitopes with high relevance for immunotherapeutic strategies in cancer.

No MeSH data available.


Characterization of phosphorylation on eluted HLA peptides.Percentage of phospho-HLA peptides commonly detected in two or more patients (a) as well as percentage of known phosphorylation sites deposited on the PhosphoSitePlus database (b). Percentage of defined amino acids affected by phosphorylation within the phosphopeptide ligandome (c). Position of phosphorylation within the eluted phospho-HLA peptides according to the peptide length, from 9 mer to 12 mer peptides (d). Logo plots of residue frequency at each position of phospho-HLA peptides according to their length (e).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5121339&req=5

f3: Characterization of phosphorylation on eluted HLA peptides.Percentage of phospho-HLA peptides commonly detected in two or more patients (a) as well as percentage of known phosphorylation sites deposited on the PhosphoSitePlus database (b). Percentage of defined amino acids affected by phosphorylation within the phosphopeptide ligandome (c). Position of phosphorylation within the eluted phospho-HLA peptides according to the peptide length, from 9 mer to 12 mer peptides (d). Logo plots of residue frequency at each position of phospho-HLA peptides according to their length (e).

Mentions: We performed a database search by enabling phosphorylation as a variable modification and although we did not specifically enrich for them, we detected a substantial fraction of phospho-HLA peptides within the eluted immunopeptidome. We filtered the list of identified phospho-HLA peptides by restricting the delta score to >15 and the localization probabilities to >0.75. After applying such stringent filters, we identified 365 phospho-HLA-I and 25 phospho-HLA-II peptides (Supplementary Data 4). About a quarter of the phospho-HLA binding peptides are shared among at least two patients and 6% of the phospho-HLA peptides have been identified independently in four or more patients' tumour samples (Fig. 3a). One third of the sites have not been previously described in the PhosphoSitePlus database30 (Fig. 3b). Additional relevant information with respect to these sites is provided in Supplementary Data 4. We observed phosphorylation in 78% of the peptides on Serine and in 19% of the peptides on Threonine. The remaining 3% are on Tyrosine (Fig. 3c). To independently check the accuracy of identification of the phospho-HLA peptides, we synthesized 10 of them and all produced identical MS-fragmentation patterns as compared with the patient derived peptides (Supplementary Figs 2–11). To determine the position of these phosphorylations, HLA-I peptides were grouped according to their length, as presented in Fig. 3d. Interestingly, independent of the broad spectrum of HLA allotypes, the modification is most prominent on the fourth position of 9–11 mer HLA-I peptides and on the fourth and sixth positions of 12 mer peptides (Fig. 3d). Moreover, we observed a preferential usage of Arginine and Lysine on position 1 (Fig. 3e) as previously reported for phospho-HLA peptides3132. Of note, a clear signature of proline-directed phosphorylation is apparent in the sequence logos of the phospho-HLA binding peptides (Fig. 3e) as was reported before32. These features therefore seem to be rather HLA allotype-independent and make them attractive to be tested as common target antigen candidates in a broader patient population. Taken altogether, our direct approach provides data about a large melanoma-associated phosphopeptide ligandome potentially attractive for targeted immunotherapies.


Direct identification of clinically relevant neoepitopes presented on native human melanoma tissue by mass spectrometry
Characterization of phosphorylation on eluted HLA peptides.Percentage of phospho-HLA peptides commonly detected in two or more patients (a) as well as percentage of known phosphorylation sites deposited on the PhosphoSitePlus database (b). Percentage of defined amino acids affected by phosphorylation within the phosphopeptide ligandome (c). Position of phosphorylation within the eluted phospho-HLA peptides according to the peptide length, from 9 mer to 12 mer peptides (d). Logo plots of residue frequency at each position of phospho-HLA peptides according to their length (e).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5121339&req=5

f3: Characterization of phosphorylation on eluted HLA peptides.Percentage of phospho-HLA peptides commonly detected in two or more patients (a) as well as percentage of known phosphorylation sites deposited on the PhosphoSitePlus database (b). Percentage of defined amino acids affected by phosphorylation within the phosphopeptide ligandome (c). Position of phosphorylation within the eluted phospho-HLA peptides according to the peptide length, from 9 mer to 12 mer peptides (d). Logo plots of residue frequency at each position of phospho-HLA peptides according to their length (e).
Mentions: We performed a database search by enabling phosphorylation as a variable modification and although we did not specifically enrich for them, we detected a substantial fraction of phospho-HLA peptides within the eluted immunopeptidome. We filtered the list of identified phospho-HLA peptides by restricting the delta score to >15 and the localization probabilities to >0.75. After applying such stringent filters, we identified 365 phospho-HLA-I and 25 phospho-HLA-II peptides (Supplementary Data 4). About a quarter of the phospho-HLA binding peptides are shared among at least two patients and 6% of the phospho-HLA peptides have been identified independently in four or more patients' tumour samples (Fig. 3a). One third of the sites have not been previously described in the PhosphoSitePlus database30 (Fig. 3b). Additional relevant information with respect to these sites is provided in Supplementary Data 4. We observed phosphorylation in 78% of the peptides on Serine and in 19% of the peptides on Threonine. The remaining 3% are on Tyrosine (Fig. 3c). To independently check the accuracy of identification of the phospho-HLA peptides, we synthesized 10 of them and all produced identical MS-fragmentation patterns as compared with the patient derived peptides (Supplementary Figs 2–11). To determine the position of these phosphorylations, HLA-I peptides were grouped according to their length, as presented in Fig. 3d. Interestingly, independent of the broad spectrum of HLA allotypes, the modification is most prominent on the fourth position of 9–11 mer HLA-I peptides and on the fourth and sixth positions of 12 mer peptides (Fig. 3d). Moreover, we observed a preferential usage of Arginine and Lysine on position 1 (Fig. 3e) as previously reported for phospho-HLA peptides3132. Of note, a clear signature of proline-directed phosphorylation is apparent in the sequence logos of the phospho-HLA binding peptides (Fig. 3e) as was reported before32. These features therefore seem to be rather HLA allotype-independent and make them attractive to be tested as common target antigen candidates in a broader patient population. Taken altogether, our direct approach provides data about a large melanoma-associated phosphopeptide ligandome potentially attractive for targeted immunotherapies.

View Article: PubMed Central - PubMed

ABSTRACT

Although mutations may represent attractive targets for immunotherapy, direct identification of mutated peptide ligands isolated from human leucocyte antigens (HLA) on the surface of native tumour tissue has so far not been successful. Using advanced mass spectrometry (MS) analysis, we survey the melanoma-associated immunopeptidome to a depth of 95,500 patient-presented peptides. We thereby discover a large spectrum of attractive target antigen candidates including cancer testis antigens and phosphopeptides. Most importantly, we identify peptide ligands presented on native tumour tissue samples harbouring somatic mutations. Four of eleven mutated ligands prove to be immunogenic by neoantigen-specific T-cell responses. Moreover, tumour-reactive T cells with specificity for selected neoantigens identified by MS are detected in the patient's tumour and peripheral blood. We conclude that direct identification of mutated peptide ligands from primary tumour material by MS is possible and yields true neoepitopes with high relevance for immunotherapeutic strategies in cancer.

No MeSH data available.